EML4-ALK fusion gene

ABSTRACT

The present inventors found that a fusion gene present in some cancer patients is an oncogene. The present invention relates to a polypeptide as a novel fusion protein, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a transformed cell comprising the vector, a method for detecting the fusion protein or polynucleotide, a method for screening a therapeutic agent for cancer, and a method for treating cancer that is shown to be positive for the fusion gene. Further, the present invention relates kit, primer set, and probe useful in the detection of cancer that is shown to be positive for the fusion gene.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a polypeptide as a novel fusionprotein, a polynucleotide encoding the polypeptide, a vector comprisingthe polynucleotide, a transformed cell comprising the vector, a methodfor detecting the fusion protein or polynucleotide, a method forscreening a therapeutic agent for cancer, and a therapeutic agent forcancer.

2. Background Art

(Carcinogenesis and Gene)

Several cancer-related genes have been known so far. In particular,tyrosine kinase genes, which encode important enzymes directlyregulating cell growth, have been known to be activated even bysubstitution or deletion in amino acid sequences and thereby bring aboutcarcinogenesis (Non-Patent Document 1).

For example, BCR-ABL fusion genes are found in most of patients withchronic myeloid leukemia. Proteins produced by this abnormal gene causethe abnormal growth of leukemia cells and simultaneously tend to inhibitblood cell apoptosis, leading to the onset of chronic myeloid leukemia(Non-Patent Document 24). Imatinib mesylate, an inhibitor of ABLtyrosine kinase, is effective for the treatment of this disease.Alternatively, TEL-JAK2 fusion proteins have been reported to beobserved in acute lymphoblastic leukemia, while NPM-ALK fusion genesencoding NPM fused with ALK tyrosine kinase are observed in more thanhalf of the cases of anaplastic large cell lymphoma (ALCL) and theactivation of ALK kinase has been shown to be important for tumor cellgrowth by NPM-ALK (Non-Patent Documents 25 and 14).

(Lung Cancer and Oncogene)

In 2004, Paez et al. (Non-Patent Document 2) and Lynch et al.(Non-Patent Document 3) have shown that epidermal growth factor receptor(EGFR) genes having sequence abnormalities are expressed in some lungcancer cells. They have also reported that gefitinib (trademark:Iressa), a kinase activity inhibitor of EGFR, is therapeuticallyeffective for patients having these EGFR mutations. Subsequent analyseshave demonstrated that EGFR mutations are frequently observed in Asians,nonsmokers, and female patients with lung cancer, and that gefitinib issignificantly effective for some of these cases (Non-Patent Documents 4and 5).

Regarding the involvement of tumor suppressor genes, it has previouslybeen reported that the inactivation of TP53 gene and Rb pathway occursin lung cancer with a high frequency (Non-Patent Document 6). Bycontrast, regarding an active type of oncogene that strongly positivelyinduces the cell growth of lung cancer, only KRAS1 gene activation insome cases has been reported (Non-Patent Document 7). The presence of anew type of abnormal kinase has been reported to be found inapproximately 10% of lung cancer cases, and this report, however, hasmade no reference to specific molecules (Non-Patent Document 36).

In 2000, EML4 (echinoderm microtubule-associated protein like protein 4)(Non-Patent Document 26) has been reported as a cytoplasmic protein witha molecular weight of 120,000, which is highly expressed in the M phaseof the cell cycle (Non-Patent Document 8). A human EML4 gene encodes apolypeptide with 981 amino acids and has 23 exons. This gene has beenmapped to chromosome 2. The EML4 protein has a basic region at the aminoterminus, as with other members of the EML family, and further hascarboxyl-terminal WD domains. The physiological functions of EML4 havebeen little known. However, according to a recent report, EML4participates in microtubule formation (Non-Patent Document 9).

On the other hand, ALK (Anaplastic Lymphoma Kinase) (Non-Patent Document27) is receptor tyrosine kinase. This protein has a transmembrane domainin the central part and has a carboxyl-terminal tyrosine kinase regionand an amino-terminal extracellular domain (Non-Patent Document 28). TheALK gene, which has 30 exons encoding a polypeptide with 1620 aminoacids, has been mapped to chromosome 2. This ALK gene has been thought,from the site or timing of its expression, to participate in thedevelopment or functions of the nervous system (Non-Patent Document 10).Loren et al. have reported from the homolog analysis of Drosophila ALKthat ALK participates in muscle differentiation (Non-Patent Document11). However, no abnormality has been observed in ALK knockout mice, andits distinct physiological functions still remain to be elucidated(Non-Patent Document 12).

Full-length ALK expression has been reported so far in some cancer cellsof ectodermal origin, such as neuroblastoma, glioblastoma, breastcancer, and melanoma (the full-length ALK expression has not beenobserved in cancer cells of endodermal and mesodermal origins)(Non-Patent Document 13). Full-length ALK is expressed in manyneuroblastoma cell lines. However, the autophosphorylation of ALK is notobserved in these neuroblastoma cell lines. Moreover, ALK expression hasbeen reported, from the cohort analysis of neuroblastoma patients, to beweakly associated with canceration. It has been suggested that ALKexpression in neuroblastoma may reflect its expression in normal neuraldifferentiation, rather than its association with canceration(Non-Patent Document 10). On the other hand, in reported cases, ligandssuch as pleiotrophin and midkine as well as the gene amplification ofALK itself increase the autophosphorylation of ALK and mobilizeintracellular signals. It has also been reported that ALK may contributeto cancer cell growth (Non-Patent Document 12).

In some cases of human malignant lymphoma and inflammatorymyofibroblastic tumor, the ALK gene has been reported to be fused withother genes (NPM, CLTCL, TFG, CARS, SEC31L1, etc.) as a result ofchromosomal translocation or inversion and thereby form a fusion type oftyrosine kinase (Non-Patent Documents 14 to 19 and 29 to 33). Moreover,a method for identifying a protein as a fusion partner for ALK using ALKantibodies has been reported (Non-Patent Document 35). On the otherhand, a fusion gene of EML4 and ALK has not been reported. Theintracellular localization of these ALK fusion proteins depends on afusion partner molecule for ALK, and the ALK fusion proteins have beenknown to exist in cytoplasm, nucleus, and the like. Since most partnermolecules have a complex formation domain, the fusion protein itself hasbeen thought to form a complex. This complex formation has beenconsidered to cause loss of control of the tyrosine kinase activity ofALK and induce carcinogenesis with abnormally activated intracellularsignals (Non-Patent Document 10). Indeed, it has been reported that theuse of ALK shRNA or ALK kinase-inhibiting compound for lymphoma cellsexpressing ALK fusion proteins can induce cell growth inhibition andcell death. Therefore, it has been suggested that the ALK fusion proteinmay serve as a therapeutic target for lymphoma and inflammatorymyofibroblastic tumor (Non-Patent Documents 20 to 22). It has also beensuggested that ALK may serve as a therapeutic target for other cancerswhose growth involves ALK as described above (Non-Patent Documents 21 to22).

Various low-molecular-weight compounds having an inhibitory activityagainst ALK have been reported so far. Marzec et al. have reported thatWHI-P131 and WHI-P154 (both, EMD Biosciences), which have originallybeen utilized as JAK3 tyrosine kinase-inhibiting substances, inhibit theactivity of NPM-ALK (Non-Patent Document 22). Another group hasdeveloped their own low-molecular-weight ALK-inhibiting substance andhas demonstrated that this inhibitor induces the cell death ofNPM-ALK-expressing lymphoma cell lines (Non-Patent Document 21). Inaddition, plural low-molecular-weight compounds having an inhibitoryactivity against ALK have been reported so far (Non-Patent Documents 23and 34 and Patent Documents 1 to 4).

[Patent Document 1] Pamphlet of WO 2005/097765

[Patent Document 2] Pamphlet of WO 2005/009389

[Patent Document 3] Pamphlet of WO 2005/016894

[Patent Document 4] Pamphlet of WO 2004/080980

[Non-Patent Document 1] “The New England journal of medicine”, (US),2005, Vol. 353, p. 172-187

[Non-Patent Document 2] “Science”, (US), 2004, Vol. 304, p. 1497-1500

[Non-Patent Document 3] “The New England journal of medicine”, (US),2004, Vol. 350, p. 2129-2139

[Non-Patent Document 4] “Cancer research”, (US), 2004, Vol. 64, p.8919-8923

[Non-Patent Document 5] “Proceedings of the national academy of sciencesof the United States of America”, (US), 2004, Vol. 101, p. 13306-13311

[Non-Patent Document 6] “Annual review of medicine”, (US), 2003, Vol.54, p. 73-87

[Non-Patent Document 7] “Seminars in oncology”, (US), 1993, Vol. 20, p.105-127

[Non-Patent Document 8] “Genomics”, (US), 2000, Vol. 68, p. 348-350

[Non-Patent Document 9] “Experimental cell research”, (US), 2006, doi:10.1016/j.yexcr.2006.06.035

[Non-Patent Document 10] “Cellular and molecular life sciences”,(Switzerland), 2004, Vol. 61, p. 2939-2953

[Non-Patent Document 11] “EMBO reports”, (UK), 2003, Vol. 4, p. 781-786

[Non-Patent Document 12] “Journal of cellular physiology”, (US), 2004,Vol. 199, p. 330-358

[Non-Patent Document 13] “International journal of cancer”, (US), 2002,Vol. 100, p. 49-56

[Non-Patent Document 14] “Science”, (US), 1994, Vol. 263, p. 1281-1284

[Non-Patent Document 15] “Blood”, (US), 1995, Vol. 86, p. 1954-1960

[Non-Patent Document 16] “Blood”, (US), 2000, Vol. 95, p. 3204-3207

[Non-Patent Document 17] “Blood”, (US), 1999, Vol. 94, p. 3265-3268

[Non-Patent Document 18] “Laboratory investigation; a journal oftechnical methods and pathology”, (US), 2003, Vol. 83, p. 1255-1265

[Non-Patent Document 19] “International journal of cancer”, (US), 2006,Vol. 118, p. 1181-1186

[Non-Patent Document 20] “Blood”, (US), 2006, Vol. 107, p. 689-697

[Non-Patent Document 21] “Blood”, (US), 2006, Vol. 107, p. 1617-1623

[Non-Patent Document 22] “Laboratory investigation; a journal oftechnical methods and pathology”, (US), 2005, Vol. 85, p. 1544-1554

[Non-Patent Document 23] “Journal of medicinal chemistry”, (US), 2006,Vol. 49, p. 1006-1015

[Non-Patent Document 24] “Cellular and molecular life sciences”,(Switzerland), 2004, Vol. 61, p. 2897-2911

[Non-Patent Document 25] “Science”, (US), 1997, Vol. 278, p. 1309-1312

[Non-Patent Document 26] GenBank accession Number: NM_(—)019063

[Non-Patent Document 27] GenBank accession Number: AB209477

[Non-Patent Document 28] Oncogene. 1997 Jan. 30; 14 (4): 439-49

[Non-Patent Document 29] Oncogene 9: 1567-1574, 1994

[Non-Patent Document 30] Am J Pathol 160: 1487-1494, 2002

[Non-Patent Document 31] Am J Pathol 157: 377-384, 2000

[Non-Patent Document 32] Blood 90: 2901-2910, 1997

[Non-Patent Document 33] Am J Pathol. 2000 March; 156 (3): 781-9

[Non-Patent Document 34] J Comb Chem. 8: 401-409, 2006

[Non-Patent Document 35] PNAS 2006 103, 7402-7407

An object of the present invention is to elucidate a polynucleotide as anovel oncogene and thereby provide a method and kit for detecting thepolynucleotide, a method for screening a therapeutic agent for cancer, amethod for treating cancer, and a therapeutic agent for cancer.

SUMMARY OF THE INVENTION

The present inventors successfully isolated, from samples obtained fromlung cancer patients, the cDNA and genomic DNA of a novel fusionpolynucleotide of an EML4 gene fused with an ALK gene as kinase(EML4-ALK fusion polynucleotide variant 1; hereinafter, referred to asan EML4-ALK fusion polynucleotide v1), which is produced by chromosomalinversion (Examples 1, 2, and 4(1)). The present inventors alsosuccessfully isolated the cDNA and genomic DNA of a novel fusionpolynucleotide (EML4-ALK fusion polynucleotide variant 2; hereinafter,referred to as an EML4-ALK fusion polynucleotide v2) whose fused regionsare different from those in the EML4-ALK fusion polynucleotide v1(Examples 4(1) and 3(3)). Analysis using clinical samples showed thatthe EML4-ALK fusion polynucleotide v1 or EML4-ALK fusion polynucleotidev2 is present in some lung cancer patients (approximately 5% to 10%)(Example 3). On the other hand, since the EML4-ALK fusion polynucleotideis an oncogene that exhibits tumorigenicity depending on its kinaseactivity (Example 6, 10(3)), it was revealed that the EML4-ALK fusionpolypeptide serves as a tool for screening a therapeutic agent forcancer that is shown to be positive for the fusion polynucleotide. Basedon these findings, the present inventors constructed a method fordetecting the fusion polynucleotide or fusion protein in a sampleobtained from a test subject (Examples 3, 4(2), 5(2), and 9) and,subsequently, a method for screening an inhibitor of the fusionpolynucleotide and/or the fusion polypeptide (i.e., a therapeutic agentfor cancer that is shown to be positive for the fusion polynucleotide)causative of cancer (Examples 7 and 10(2)), and confirmed that compoundsobtained by screening exhibit an anti-tumor effect (Examples 8(3), 8(7),and 8(8)). As a result, a test subject from which the fusionpolynucleotide has been detected can receive cancer treatment using theinhibitor of the fusion polynucleotide and/or the polypeptide encodedthereby. According to the detection method, subjects to which thetherapeutic agent is applicable can be selected. As a result,tailor-made medical care expected as highly effective treatment usingthe inhibitor can be carried out.

Based on these findings, the present inventors provided a novelpolynucleotide and polypeptide useful as screening tools, a screeningmethod, and a method for treating cancer that is shown to be positivefor a fusion gene of EML4 gene and ALK gene. The present inventorscompleted the present invention by further providing a detection methoduseful in the detection of cancer that is shown to be positive for thefusion gene of EML4 gene and ALK gene.

Specifically, the present invention relates to:

[1] an isolated polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 2 or 7 and having a kinase activity;

[2] the isolated polypeptide according to [1] consisting of the aminoacid sequence represented by SEQ ID NO: 2 or 7;

[3] an isolated polynucleotide encoding the polypeptide according to[1];

[4] an expression vector comprising the polynucleotide according to [3];

[5] a cell transformed with the expression vector according to [4];

[6] a method for producing the polypeptide according to [1], comprisingculturing a transformed cell according to [5] under conditions suitablefor polypeptide expression and collecting the polypeptide from the cell;

[7] a method for detecting a fusion gene of EML4 gene and ALK gene,comprising the step of detecting the presence of the polynucleotideencoding the polypeptide according to [1] in a sample obtained from atest subject;

[8] a method for detecting a fusion protein encoded by a fusion gene ofEML4 gene and ALK gene, comprising the step of detecting the presence ofthe polypeptide according to [1] in a sample obtained from a testsubject;

[9] a kit for detection of a fusion gene of EML4 gene and ALK gene,comprising sense and antisense primers designed to specifically amplifya polynucleotide encoding the polypeptide according to [1];

[10] a primer set for detecting a fusion gene of EML4 gene and ALK gene,comprising an antisense primer consisting of a nucleic acid moleculehybridizing under stringent conditions to i) the polynucleotideaccording to [3], ii) a polynucleotide consisting of the nucleotidesequence represented by SEQ ID NO: 4, and/or iii) a polynucleotideconsisting of the nucleotide sequence represented by SEQ ID NO: 5, and asense primer consisting of nucleic acid molecule hybridizing understringent conditions to complementary strands to the above i) to iii);

[11] a primer set of a sense primer comprising an oligonucleotide withat least any 16 consecutive bases located at base Nos. 1 to 1759 in SEQID NO: 1 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 1760 to 3926 in SEQ ID NO: 1, or a primer setconsisting of complementary strands thereof, wherein the sense andantisense primers give amplification products of 1 kb or less in size;

[12] the primer set of the sense primer and the antisense primer inaccording [11] selected from a group consisting of (1)-(11) below orcomplementary strands thereof; (1) SEQ ID NOs: 8 and 9, (2) SEQ ID NOs:61 and 62, (3) SEQ ID NOs: 63 and 64, (4) SEQ ID NOs: 65 and 66, (5) SEQID NOs: 67 and 68, (6) SEQ ID NOs: 69 and 70, (7) SEQ ID NOs: 71 and 72,(8) SEQ ID NOs: 73 and 74, (9) SEQ ID NOs: 75 and 76, (10) SEQ ID NOs:77 and 78, and (11) SEQ ID NOs: 79 and 80.

[13] a primer set of a sense primer comprising an oligonucleotide withat least any 16 consecutive bases located at base Nos. 1 to 2242 in SEQID NO: 6 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 2243 to 3933 in SEQ ID NO: 6, or a primer setconsisting of complementary strands thereof, wherein the sense andantisense primers give amplification products of 1 kb or less in size;

[14] the primer set of the sense primer and the antisense primer inaccording to [13] selected from a group consisting of (1)-(10) below orcomplementary strands thereof, (1) SEQ ID NOs: 81 and 82, (2) SEQ IDNOs: 83 and 84, (3) SEQ ID NOs: 85 and 86, (4) SEQ ID NOs: 87 and 88,(5) SEQ ID NOs: 89 and 90, (6) SEQ ID NOs: 91 and 92, (7) SEQ ID NOs: 93and 94, (8) SEQ ID NOs: 95 and 96, (9) SEQ ID NOs: 97 and 98, and (10)SEQ ID NOs: 99 and 100.

[15] a primer set of a sense primer comprising an oligonucleotide withat least any 16 consecutive bases located at base Nos. 1 to 3629 in SEQID NO: 4 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 3630 to 3979 in SEQ ID NO: 4, or a primer setconsisting of complementary strands thereof, wherein the sense andantisense primers give amplification products of 1 kb or less in size;

[16] the primer set of the sense primer and the antisense primer inaccording to [15] selected from a group consisting of (1)-(10) below orcomplementary strands thereof, (1) SEQ ID NOs: 15 and 16, (2) SEQ IDNOs: 17 and 18, (3) SEQ ID NOs: 19 and 20, (4) SEQ ID NOs: 21 and 22,(5) SEQ ID NOs: 23 and 24, (6) SEQ ID NOs: 25 and 26, (7) SEQ ID NOs: 27and 28, (8) SEQ ID NOs: 29 and 30, (9) SEQ ID NOs: 31 and 32, and (10)SEQ ID NOs: 33 and 34.

[17] a primer set of a sense primer comprising an oligonucleotide withat least any 16 consecutive bases located at base Nos. 1 to 579 in SEQID NO: 5 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 580 to 853 in SEQ ID NO: 5, or a primer setconsisting of complementary strands thereof,

[18] the primer set of the sense primer and the antisense primer inaccording to [17] selected from a group consisting of (1)-(11) below orcomplementary strands thereof, (1) SEQ ID NOs: 35 and 36, (2) SEQ IDNOs: 37 and 38, (3) SEQ ID NOs: 39 and 18, (4) SEQ ID NOs: 41 and 20,(5) SEQ ID NOs: 43 and 22, (6) SEQ ID NOs: 45 and 46, (7) SEQ ID NOs: 47and 26, (8) SEQ ID NOs: 49 and 28, (9) SEQ ID NOs: 51 and 52, (10) SEQID NOs: 53 and 54, and (11) SEQ ID NOs: 55 and 34.

[19] a probe for detecting the polynucleotide of the present invention,comprising a nucleic acid molecule with at least 32 consecutive baseshybridizing under stringent conditions to i) the polynucleotideaccording to [3], ii) a polynucleotide consisting of the nucleotidesequence represented by SEQ ID NO: 4, iii) a polynucleotide consistingof the nucleotide sequence represented by SEQ ID NO: 5, or iv)complementary strands to the above i) to iii), and comprising positions1744 to 1775 of the nucleotide sequence represented by SEQ ID NO: 1,positions 2227 to 2258 of the nucleotide sequence represented by SEQ IDNO: 6, positions 3614 to 3645 of the nucleotide sequence represented bySEQ ID NO: 4, positions 564 to 595 of the nucleotide sequencerepresented by SEQ ID NO: 5; or complementary strands thereof.

[20] a method for screening a substance inhibiting the polypeptideaccording to [1], comprising the steps of (1) bringing test substancesinto contact with the polypeptide or a cell expressing the polypeptide,(2) analyzing whether the polypeptide is inhibited or not, and (3)selecting a substance inhibiting the polypeptide;

[21] the screening method according to [20], further comprising the stepof confirming that the selected test substance has a therapeuticactivity against cancer that is shown to be positive for a fusion geneof EML4 gene and ALK gene;

[22] a method for treating cancer that is shown to be positive for afusion gene of EML4 gene and ALK gene, comprising administering aneffective amount of a substance inhibiting the polypeptide according to[1] to a target in need of treatment of cancer that is shown to bepositive for a fusion gene of EML4 gene and ALK gene;

[23] the method for treating cancer that is shown to be positive for afusion gene of EML4 gene and ALK gene according to [22], wherein thesubstance inhibiting the polypeptide according to [1] is5-chloro-N⁴-[2-(isopropylsulfonyl)phenyl]-N²-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4-diamineor2-[(5-bromo-2-{[2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide;

[24] a double-stranded nucleic acid having an inhibitory activityagainst the expression of the polypeptide according to [1], wherein adouble-stranded portion is designed on the basis of bases at positionsselected from a group consisting of (1)1743 to 1761, (2)1744 to 1762,(3)1750 to 1768, (4)1753 to 1771, (5)1756 to 1774, and (6)1757 to 1775in SEQ ID NO: 1; and

[25] a method for treating cancer that is shown to be positive for afusion gene of EML4 gene and ALK gene, comprising administering aneffective amount of the double-stranded nucleic acid according to [24]to a subject in need of treatment of cancer that is shown to be positivefor a fusion gene of EML4 gene and ALK gene.

None of above-mentioned documents have reported the formation of afusion gene by EML4 gene and ALK gene, let alone the expression of thefusion gene of EML4 gene and ALK gene in some cancer patients. Theformation of a fusion gene by EML4 gene and ALK gene and the expressionof this fusion gene in some cancer patients were found for the firsttime by the present inventors. The screening method using a fusion geneof EML4 gene and ALK gene is an invention that was made for the firsttime by the present inventors. Moreover, the method for detecting thefusion gene useful in the detection of cancer that is shown to bepositive for a fusion gene, the probe or primer set useful in thisdetection, and the kit for detection are inventions that were providedfor the first time by the findings of the present inventors. Various ALKinhibitors (Patent Documents 3 to 4 and Non-Patent Documents 20 to 22and 34) including5-chloro-N⁴-[2-(isopropylsulfonyl)phenyl]-N²-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4-diamineand2-[(5-bromo-2-{[2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzenesulfonamidehave been reported. Furthermore ALK inhibitors have been reported toinduce growth inhibition and cell death in lymphoma cells expressingNPM-ALK fusion proteins (Non-Patent Documents 20 to 22). However, it hastotally been unknown that these ALK inhibitors have therapeuticapplications for cancer (particularly, lung cancer) that is shown to bepositive for a fusion gene of EML4 gene and ALK gene. The method fortreating cancer (particularly, lung cancer) that is shown to be positivefor a fusion gene of EML4 gene and ALK gene is an invention that wasprovided for the first time by the findings of the present inventors. ANPM-ALK inhibitor which suppressed lymphomagenesis in ALK-positive ALCLhas been reported (PNAS, 2007, Jan. 2, 104 (1), 270-275 Epub2006December). However this PNAS article is the literature published withinone year prior to the date of the present application. The presence of anew type of abnormal kinase found in approximately 10% of lung cancercases have been reported (Proceedings of the 65th Annual Meeting of theJapanese Cancer Association, O-324 (issued on Aug. 28, 2006)). EML4-ALKfusion gene and its transforming activity have been reported (Nature448, 561-566, 2 August 2007 (online on 11 Jul. 2007)). Further, in theNature article, it has been described that the fusion kinase is apromising candidate for therapeutic target as well as for a diagnosticmolecular marker in non-small-cell lung cancer. However, above O-324article and the Nature article are the literature published within oneyear prior to the date of the present application.

The polypeptide, polynucleotide, expression vector, and cell of thepresent invention can be used in the screening of a substance inhibitingthe polypeptide of the present invention (particularly, a therapeuticagent for lung cancer that is shown to be positive for a fusion gene ofEML4 gene and ALK gene). Subjects for which a fusion gene of EML4 geneand ALK gene is positive (particularly, lung cancer patients) can bedetected by using the presence of the polypeptide and/or polynucleotideof the present invention as an index. According to the screening methodof the present invention, a therapeutic agent for cancer (particularly,a therapeutic agent for lung cancer) that is shown to be positive for afusion gene of EML4 gene and ALK gene can be screened. The probe orprimer and kit for detection of the present invention can be utilizedfor detecting a cancer that is shown to be positive for a fusion gene ofEML4 gene and ALK gene. The detection method of the present inventioncan be utilized as a method for detecting cancer (particularly, lungcancer) that is shown to be positive for the fusion gene of EML4 geneand ALK gene. Moreover, according to the detection method of the presentinvention, whether or not the therapeutic agent of the present inventionis applicable to subjects can be determined. The substance inhibitingthe polypeptide of the present invention is useful as a therapeuticagent for cancer, particularly lung cancer, that is shown to be positivefor a fusion gene of EML4 gene and ALK gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the results of PCR. The left lane (46, XX) shows the resultwhen the genomic DNA from a normal healthy subject was used as asubstrate, and the right lane (ID #33) shows the result when the genomicDNA from a cancer patient was used as a substrate;

FIG. 2 shows the results of the screening for EML4-ALK fusionpolynucleotide in specimens of lung cancer patients. Lane “46, XX” showsthe result of using peripheral monocytes of a normal healthy femalesubject, and “ID #2” to “ID #42” show the result of using samplesobtained from excised specimens from lung cancer patients. In addition,lane “NTC” shows the result without added substrate cDNA. Lane “marker”is the lane where the size marker DNA was electrophoresed (uppersection). The results of amplification of GAPDH cDNA are shown in thelower section. Sex (M, male; F, female), pathology (S, squamous cellcarcinoma; A, adenocarcinoma; AS, adenosquamous carcinoma; B,bronchiolo-aleveolar carcinoma) and the presence or absence of EGFRmutation and the presence or absence of smoking history are shown in theupper part of the figure;

FIG. 3 shows tumorgenicity of the genes. The upper section of the figure(3T3) shows 3T3 fibroblast cells when a blank vector (Vector), andexpression plasmid such as full length ALK/pMXS (ALK), EML4-ALKv1/pMXS(EML4-ALK) or EML4-ALK (K589M)/pMXS were introduced. The scale barrepresents 100 μm. The lower section of the figure (Nude mice) shows theresult of the inoculation of each 3T3 fibroblast cell line to nude mice;

FIG. 4 shows the inhibitory effect of a EML4-ALK fusion polypeptideinhibitor (compound A) on intracellular autophosphorylation. “KM”indicates when EML4-ALK (K589M) expressing cells were used, and “EA”shows when v1 expressing BA/F3 cells were used. “αp-ALK” (upper panel)shows the result of the immunoblotting when anti-phosphorylated ALKantibody was used, and “αFLAG” (lower panel) shows the result of theimmunoblotting when anti-FLAG antibody was used;

FIG. 5 shows the growth potential of cells which express CD8 proteinonly (CD8), or co-express CD8 and ALK (ALK), CD8 and EML4-ALK fusionpolypeptide v1 (EA) or CD8 and EML4-ALK (K589M) (KM) in the presence(+IL-3) or absence (−IL-3) of IL-3. The horizontal axis of the figure istime course (Days) and the vertical axis is the cell number;

FIG. 6( a) shows time dependent change of cell number when respectiveconcentrations of compound A were added to BA/F3 cells expressing onlyCD8 and cultured in the presence of IL-3. (b) shows time dependentchange of cell number when v1 expressing BA/F3 cells were cultured withrespective concentration of compound A in the absence of IL-3. Thehorizontal axis of the figure is time course (Days) and the verticalaxis is the cell number; and

FIG. 7 shows siRNA1-siRNA9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in detail. Genemanipulation techniques described herein can be practiced according totechniques known in the art, such as “Molecular Cloning”, Sambrook, J etal., Cold Spring Harbor Laboratory Press, 1989, unless otherwisespecified. Protein manipulation techniques described herein can bepracticed according to techniques known in the art, such as“Experimental Protocol on Proteins”, Shujunsha Co. Ltd. 1997, unlessotherwise specified.

The phrase “the polypeptide of the present invention is inhibited”described herein encompasses both the phrases “the expression of thepolypeptide of the present invention is inhibited” and “the activity ofthe polypeptide of the present invention is inhibited”. A “substanceinhibiting the polypeptide of the present invention” encompasses both a“substance inhibiting the expression of the polypeptide of the presentinvention” and a “substance inhibiting the activity of the polypeptideof the present invention”.

<Polypeptide, Polynucleotide, Expression Vector, Transformed Cell, andMethods for Producing Polypeptide of the Present Invention>

The polypeptide of the present invention encompasses:

(1) a polypeptide consisting of the amino acid sequence represented bySEQ ID NO: 2 or 7; and

(2) (a) a polypeptide comprising the amino acid sequence represented bySEQ ID NO: 2 and having a kinase activity (hereinafter, referred to as av1 functionally equivalent modified polypeptide);

(b) a polypeptide comprising the amino acid sequence represented by SEQID NO: 7 and having a kinase activity (hereinafter, referred to as a v2functionally equivalent modified polypeptide);

(hereinafter, the v1 functionally equivalent modified polypeptide and v2functionally equivalent modified polypeptide are collectively referredto as a functionally equivalent modified polypeptide).

The phrase “having a kinase activity” means having an activity as anenzyme phosphorylating tyrosine. Whether a certain polypeptide “has akinase activity” is confirmed by a method of Example 7(2).

Up to this point, the polypeptide of the present invention has beendescribed. Hereinafter, the polypeptide consisting of the amino acidsequence represented by SEQ ID NO: 2 or 7, and the functionallyequivalent modified polypeptide are collectively referred to as a“polypeptide of the present invention”. Of the polypeptides of thepresent invention, the polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO: 2, and the v1 functionally equivalent modifiedpolypeptide are collectively referred to as a “polypeptide type v1 ofthe present invention”. The polypeptide consisting of the amino acidsequence represented by SEQ ID NO: 7, and the v2 functionally equivalentmodified polypeptide are collectively referred to as a “polypeptide typev2 of the present invention”. A protein as the polypeptide consisting ofthe amino acid sequence represented by SEQ ID NO: 2 is referred to as an“EML4-ALK fusion polypeptide v1”. A protein as the polypeptideconsisting of the amino acid sequence represented by SEQ ID NO: 7 isreferred to as “an EML4-ALK fusion polypeptide v2”. The “EML4-ALK fusionpolypeptide v1” and the “EML4-ALK fusion polypeptide v2” arecollectively referred to as an “EML4-ALK fusion polypeptide”.

The polypeptide of the present invention is, preferably, the“polypeptide consisting of the amino acid sequence represented by SEQ IDNO: 2 or 7”, or the “polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 2 or 7 and having a kinase activity”, morepreferably, the “polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO: 2 or 7”.

A polynucleotide encoding the polypeptide of the present invention(hereinafter, referred to as a “polynucleotide of the presentinvention”) is a polynucleotide represented by a nucleotide sequenceencoding the EML4-ALK fusion polypeptide, or functionally equivalentmodified polypeptide. The polynucleotide of the present invention is,preferably, a polynucleotide consisting of a nucleotide sequenceencoding the amino acid sequence represented by SEQ ID NO: 2 or 7, morepreferably, a polynucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 1 (particularly preferably, positions 271 to3447 in SEQ ID NO: 1) or 6.

Of the polynucleotides of the present invention, a gene encoding thepolypeptide type v1 of the present invention is referred to as a“polynucleotide type v1 of the present invention”. A gene encoding thepolypeptide type v2 of the present invention is referred to as a“polynucleotide type v2 of the present invention”.

A “fusion gene of EML4 gene and ALK gene” described herein refers to thepolynucleotide of the present invention. A gene encoding the EML4-ALKfusion polypeptide v1, which is a polynucleotide type v1 of the presentinvention, is referred to as an “EML4-ALK fusion polynucleotide v1”. Agene encoding the EML4-ALK fusion polypeptide v2, which is apolynucleotide type v2 of the present invention, is referred to as an“EML4-ALK fusion polynucleotide v2”. The EML4-ALK fusion polynucleotidesv1 and v2 are collectively referred to as an “EML4-ALK fusionpolynucleotide”.

The phrase “cancer that is shown to be positive for a fusion gene ofEML4 gene and ALK gene” described herein means cancer positive for thepolynucleotide of the present invention (i.e., the polynucleotide of thepresent invention is present) and, preferably, means cancer positive forthe EML4-ALK fusion polynucleotide (i.e., the EML4-ALK fusionpolynucleotide is present). Among “cancer that is shown to be positivefor a fusion gene of EML4 gene and ALK gene” described herein, theEML4-ALK fusion polynucleotide-positive cancer is preferable.

Examples of a method for producing the polynucleotide of the presentinvention can include, but not particularly limited to, (1) a methodusing polymerase chain reaction (PCR), (2) a method using a standardgenetic engineering approach (i.e., a method comprising selecting atransformed strain comprising desired amino acid sequence from strainstransformed with a cDNA library), and (3) a chemical synthesis method.Each production method can be practiced in the same way as in WO01/34785. However, the “novel protein of the present invention”described therein can be interpreted as a protein consisting of thepolypeptide of the present invention described herein, and the “gene ofthe present invention” described therein can be interpreted as thepolynucleotide of the present invention described herein.

In the method using PCR, the polynucleotide of the present invention canbe produced, for example, according to procedures described in 1)Production method of protein gene, a) First production method in“Embodiments of the Invention” of the patent document. mRNA is extractedfrom cells or tissues having the ability to produce the protein of thepresent invention, for example, from lung tissues derived from a humanpatient with lung cancer. Subsequently, this mRNA can be subjected toreverse transcriptase reaction in the presence of random primers oroligo dT primers to synthesize single-stranded cDNA. The obtainedsingle-stranded cDNA can be subjected to PCR using 2 primers interposinga partial region of the gene of interest to obtain the polynucleotide ofthe present invention or a portion thereof. More specifically, thepolynucleotide of the present invention can be produced, for example, bya method described in Example 4(1).

Alternatively, the polynucleotide of the present invention may beproduced by artificially synthesizing the polynucleotide of the presentinvention as separated fragments by reverse transcription (RT)-PCR andthen fusing these obtained fragments. For example, (a) 1489 baseslocated from the initiation codon (ATG) of exon 1 to exon 13 in EML4(for the polynucleotide type v1 of the present invention) or 2242 baseslocated from the initiation codon of exon 1 to exon 20 in EML4 (for thepolynucleotide type v2 of the present invention) are amplified by RT-PCRusing, as a template, mRNA extracted from cells (e.g., HeLa cells) ortissues endogenously expressing EML4 and using 2 primers interposing thegene region of interest. On the other hand, for example, (b) 1691 baseslocated from exon 21 to stop codon (TGA) in ALK (for both thepolynucleotide type v1 of the present invention and the polynucleotidetype v2 of the present invention) are amplified by RT-PCR using, as atemplate, mRNA extracted from cells (e.g., Rh30 or U-87MG cells) ortissues endogenously expressing ALK and using 2 primers interposing thegene region of interest. The amplified PCR products of (a) and (b) canbe fused to obtain the polynucleotide of the present invention. Thisfusion can be practiced by devising the primers used in the RT-PCR of(a) and (b). For example, a primer is created by adding approximately 10bases of the 5′-terminal antisense nucleotide sequence of exon 21 in ALKto the 5′ terminus of an antisense primer for the RT-PCR amplificationof the fragment of (a), and this created primer is used as an antisenseprimer to amplify the fragment of (a). Moreover, a primer is created byadding approximately 10 bases of the 3′-terminal sense nucleotidesequence of exon 13 in EML4 (for the polynucleotide type v1 of thepresent invention) or approximately 10 bases of the 3′-terminal sensenucleotide sequence of exon 20 in EML4 (for the polynucleotide type v2of the present invention) to the 5′ terminus of a sense primer for theRT-PCR amplification of the fragment of (b), and this created primer isused as a sense primer to amplify the fragment of (b). Thepolynucleotide of the present invention can be obtained by PCR using, astemplates, these 2 kinds of PCR products obtained and using a senseprimer comprising the initiation codon of EML4 and an antisense primercomprising the stop codon of ALK. Alternatively, the polynucleotide ofthe present invention can also be obtained by annealing and extensionreactions using only these 2 kinds of PCR products obtained withoutusing the sense primer comprising the initiation codon of EML4 and theantisense primer comprising the stop codon of ALK.

In the method using a standard genetic engineering approach, thepolynucleotide of the present invention can be produced, for example,according to procedures described in 1) Production method of proteingene, b) Second production method in “Mode for Carrying Out theInvention” of WO 01/34785.

In the method using a chemical synthesis method, the polynucleotide ofthe present invention can be produced, for example, according toprocedures described in 1) Production method of protein gene, c) Thirdproduction method and d) Fourth production method in “Mode for CarryingOut the Invention” of WO 01/34785. More specifically, the polynucleotideof the present invention can also be produced by binding nucleotidefragments produced by the chemical synthesis method. Alternatively, eachpolynucleotide oroligonucleotide can be synthesized with a DNAsynthesizer (e.g., Oligo 1000M DNA Synthesizer (Beckman) or 394 DNA/RNASynthesizer (Applied Biosystems).

The expression vector of the present invention, transformed cell of thepresent invention, and method for producing polypeptide of the presentinvention can be practiced, for example, according to proceduresdescribed in 2) Methods for the production of the vector of theinvention, the host cell of the invention and the recombinant protein ofthe invention in “Mode for Carrying Out the Invention” of WO 01/34785.The isolated polynucleotide of the present invention can be incorporatedagain into appropriate vector DNA to thereby transform a eukaryotic orprokaryotic host cell therewith. Alternatively, an appropriate promoterand a sequence involved in phenotypic expression may be introduced tothe vector to thereby cause each host cell transformed therewith toexpress the polynucleotide.

The expression vector of the present invention is not particularlylimited as long as it comprises the polynucleotide of the presentinvention and it expresses the polypeptide of the present invention.Examples thereof can include an expression vector obtained by insertingthe polynucleotide of the present invention into an expression vectorknown in the art, which is appropriately selected according to a hostcell used.

Likewise, the cell of the present invention is not particularly limitedas long as it comprises the polynucleotide of the present invention as aresult of nucleic acid transfer by transfection or infection with theexpression vector of the present invention. For example, the cell of thepresent invention can be a host cell comprising the polynucleotide ofthe present invention incorporated in the chromosome or can be a cellcomprising the expression vector comprising the polynucleotide of thepresent invention. Moreover, the original cell that undergoes nucleicacid transfer can be a cell expressing the polypeptide of the presentinvention or can be a cell not expressing the polypeptide of the presentinvention. The cell of the present invention can be obtained, forexample, by transfecting or infecting a desired cell with the expressionvector of the present invention. More specifically, for example, thepolynucleotide of the present invention can be incorporated to anexpression vector for mammalian cells to thereby obtain an expressionvector of the desired protein. This expression vector can be taken upinto a cell by use of a commercially available transfection reagentLipofectamine to thereby produce the transformed cell of the presentinvention. Alternatively, for example, the expression vector comprisingthe polynucleotide of the present invention and a plasmid for packaging(e.g., pGP or pE-eco) can be introduced into a BOSC23 cell by use of acommercially available transfection reagent Lipofectamine, as describedin Example 1, to thereby produce an expression retrovirus. A BA/F3 cellcan be infected with this retrovirus to thereby produce the transformedcell of the present invention.

The desired transformed cell thus obtained can be cultured according toa standard method. A protein consisting of the polypeptide of thepresent invention is produced by this culture. A medium used in theculture can be selected appropriately according to a host cell used fromamong a variety of routine media. For example, an RPMI1640 mediumsupplemented with serum components such as fetal bovine serum (FBS) canbe used for the BA/F3 cell.

The polypeptide of the present invention thus produced by thetransformed cell can be separated and purified by a variety ofseparation operation techniques known in the art using the physical orbiochemical properties of the polypeptide.

The polypeptide of the present invention can be fused in frame with amarker sequence and expressed to thereby achieve the confirmation ofexpression of the protein as well as the purification of the protein.Examples of the marker sequence include FLAG epitope, Hexa-Histidinetag, Hemagglutinin tag, and myc epitope. Alternatively, a specific aminoacid sequence recognizable by protease such as enterokinase, factor Xa,or thrombin can be inserted between the marker sequence and thepolypeptide of the present invention to thereby remove the markersequence portion by cleavage with the protease.

The polynucleotide of the present invention can be used as a controltemplate for reaction in the detection method using PCR and is useful inthe determination of a subject for which the polynucleotide of thepresent invention is positive. Moreover, the polypeptide of the presentinvention can be used as a control for detecting and quantifyingexpression levels.

<Probe or Primer of the Present Invention>

The present invention encompasses a probe or primers useful in thedetection of the presence of the polynucleotide of the presentinvention.

Specifically, the present invention encompasses:

(1) a primer set for detecting the polynucleotide of the presentinvention, comprising nucleic acid molecules with at least 16consecutive bases hybridizing under stringent conditions (preferably,more stringent conditions) to a polynucleotide type v1 of the presentinvention (preferably, aEML4-ALK fusion polynucleotide v1, morepreferably, a polynucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 1), a polynucleotide type v2 of the presentinvention (preferably, a EML4-ALK fusion polynucleotide v2, morepreferably, a polynucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 6), a polynucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 4, which is one ofsequences comprising the fusion point of a polynucleotide belonging toEML4-ALK fusion polynucleotide v1, and/or a polynucleotide consisting ofthe nucleotide sequence represented by SEQ ID NO: 5, which is one ofsequences comprising the fusion point of a polynucleotide belonging toEMK4-ALK fusion polynucleotide v2, or complementary strands thereof;

(2) a probe for detecting the polynucleotide of the present invention,comprising a nucleic acid molecule with at least 32 consecutive baseshybridizing under stringent conditions (preferably, more stringentconditions) to a polynucleotide type v1 of the present invention(preferably, aEML4-ALK fusion polynucleotide v1, more preferably, apolynucleotide consisting of the nucleotide sequence represented by SEQID NO: 1), a polynucleotide encoding the polypeptide type v2 of thepresent invention (preferably, aEML4-ALK fusion polynucleotide v2, morepreferably, a polynucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO: 6), a polynucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 4, which is one ofsequences comprising the fusion point of a polynucleotide belonging toEML4-ALK fusion polynucleotide v1, a polynucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 5, which is one ofsequences comprising the fusion point of a polynucleotide belonging toEMK4-ALK fusion polynucleotide v2; or complementary strands thereof, andcomprising positions 1744 to 1775 of the nucleotide sequence representedby SEQ ID NO: 1, positions 2227 to 2258 of the nucleotide sequencerepresented by SEQ ID NO: 6, positions 3614 to 3645 of the nucleotidesequence represented by SEQ ID NO: 4, positions 564 to 595 of thenucleotide sequence represented by SEQ ID NO: 5; or complementarystrands thereof (preferably, positions 1742 to 1777 of the nucleotidesequence represented by SEQ ID NO: 1, positions 2225 to 2260 of thenucleotide sequence represented by SEQ ID NO: 6, positions 3612 to 3647of the nucleotide sequence represented by SEQ ID NO: 4, or positions 562to 597 of the nucleotide sequence represented by SEQ ID NO: 5,particularly preferably, positions 1740 to 1779 of the nucleotidesequence represented by SEQ ID NO: 1, positions 2223 to 2262 of thenucleotide sequence represented by SEQ ID NO: 6, positions 3610 to 3649of the nucleotide sequence represented by SEQ ID NO: 4, or positions 560to 599 of the nucleotide sequence represented by SEQ ID NO: 5; orcomplementary strands thereof);

(3) a primer set of a sense primer comprising an oligonucleotide with atleast any 16 consecutive bases located at base Nos. 1 to 1759(preferably, base Nos. 271 to 1759) in SEQ ID NO: 1 and an antisenseprimer comprising an oligonucleotide complementary to an oligonucleotidewith at least any 16 consecutive bases located at base Nos. 1760 to 3926(preferably, base Nos. 1760 to 3447) in SEQ ID NO: 1, or a primer setconsisting of complementary strands thereof, wherein the spacing betweenthe selected positions of the sense and antisense primers in SEQ ID NO:1 is 1 kb or less, or wherein the sense and antisense primers giveamplification products of 1 kb or less in size;

(4) a primer set of a sense primer comprising an oligonucleotide with atleast any 16 consecutive bases located at base Nos. 1 to 2242 in SEQ IDNO: 6 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 2243 to 3933 in SEQ ID NO: 6, or a primer setconsisting of complementary strands thereof, wherein the spacing betweenthe selected positions of the sense and antisense primers in SEQ ID NO:6 is 1 kb or less, or wherein the sense and antisense primers giveamplification products of 1 kb or less in size;

(5) a primer set of a sense primer comprising an oligonucleotide with atleast any 16 consecutive bases located at base Nos. 1 to 3629 in SEQ IDNO: 4 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 3630 to 3979 in SEQ ID NO: 4, or a primer setconsisting of complementary strands thereof, wherein the spacing betweenthe selected positions of the sense and antisense primers in SEQ ID NO:4 is 1 kb or less, or wherein the sense and antisense primers giveamplification products of 1 kb or less in size; and

(6) a primer set of a sense primer comprising an oligonucleotide with atleast any 16 consecutive bases located at base Nos. 1 to 579 in SEQ IDNO: 5 and an antisense primer comprising an oligonucleotidecomplementary to an oligonucleotide with at least any 16 consecutivebases located at base Nos. 580 to 853 in SEQ ID NO: 5, or a primer setconsisting of complementary strands thereof. Examples of a preferableprimer set include:

(7) the primer set or the primer set consisting of complementary strandsthereof according to any of (3) to (6), wherein the sense and antisenseprimers are represented by (i) SEQ ID NOs: 8 and 9, (ii) SEQ ID NOs: 15and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and28, 29 and 30, 31 and 32, or 33 and 34, (iii) SEQ ID NOs: 35 and 36, 37and 38, 39 and 18, 41 and 20, 43 and 22, 45 and 46, 47 and 26, 49 and28, 51 and 52, 53 and 54, or 55 and 34, (iv) SEQ ID NOs: 61 and 62, 63and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and76, 77 and 78, or 79 and 80, or (v) SEQ ID NOs: 81 and 82, 83 and 84, 85and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and98, or 99 and 100.

The primer sets (7) (i) (in Example 3(1)), (7) (ii) (in Example 3(2)),(7) (iii) (in Example 3(3)), and (7) (iv) and (v) (in Example 4(2)) wereused for detecting the presence of the polynucleotide of the presentinvention.

Fusion point in this specification means a point where a portion derivedfrom EML4 gene and a portion derived from ALK gene are fused. Fusionpoint in SEQ ID No: 1 is the point where a nucleotide of base position1759 and a nucleotide of base position 1760 are fused. Fusion point inSEQ ID No: 6 is the point where a nucleotide of base position 2242 and anucleotide of base position 2243 are fused. Fusion point in SEQ ID No: 4is the point where a nucleotide of base position 3629 and a nucleotideof base position 3630 are fused. Fusion point in SEQ ID No: 5 is thepoint where a nucleotide of base position 579 and a nucleotide of baseposition 580 are fused.

The “stringent conditions” comprise hybridization conditions on theorder of “5×SSPE, 5× Denhardt's solution, 0.5% SDS, 50% formamide, 200μg/ml salmon sperm DNA, 42° C. overnight” and washing conditions on theorder of “0.5×SSC, 0.1% SDS, 42° C.”. The “more stringent conditions”comprise hybridization conditions on the order of “5×SSPE, 5× Denhardt'ssolution, 0.5% SDS, 50% formamide, 200 μg/ml salmon sperm DNA, 42° C.overnight” and washing conditions on the order of “0.2×SSC, 0.1% SDS,65° C.”.

The probe or primers of the present invention can be utilized as a probefor detecting and isolating the polynucleotide of the present inventionor as primers for amplifying the polynucleotide of the presentinvention. For primer use, its chain length is usually 15 to 40 bases,preferably 16 to 24 bases, more preferably 18 to 24 bases, particularlypreferably 20 to 24 bases. Alternatively, for probe use, the DNA is atleast 32 bases, more preferably, at least 36 bases, particularlypreferably, at least 40 bases or longer, in chain length.

Methods for producing the probe and primers of the present invention arenot particularly limited. The probe and primers of the present inventioncan be produced by the chemical synthesis method used for the method forproducing the polynucleotide of the present invention.

Based on the present invention, an array of oligonucleotide probescomprising the nucleotide sequence of the polynucleotide of the presentinvention or a fragment thereof can be constructed. The array techniqueis known in the art and has been used for analyzing gene expression(Chee, M. et al. (1996) Science, 274, 610-613).

<Detection Method and Kit for Detection of the Present Invention>

The present invention encompasses a method for detecting thepolynucleotide of the present invention and a method for detecting afusion protein consisting of the polypeptide of the present invention.The EML4-ALK fusion polynucleotide was found in some human patients withcancer (specifically, non-small cell lung cancer). Therefore, cancer(preferably, lung cancer) that is shown to be positive for thepolynucleotide of the present invention can be detected by utilizing thepresence of this fusion polynucleotide. Specifically, an aspectcomprising the step below is exemplified. Specifically, the method fordetecting the polynucleotide of the present invention comprises the stepof

(1) Detecting the presence of the polynucleotide of the presentinvention in a sample obtained from a test subject.

A sample collected from a test subject (a sample separated from thebody), specifically, any collected body fluid (preferably, blood),bronchoalveolar lavage fluid, biopsied sample, or sputum sample is usedas the sample obtained from a test subject. Preferably, a biopsy sampleof the affected part of the lung of the test subject or a sputum sampleof the test subject is used. Genomic DNA extracted from the sample or atranscription product (product as a result of transcription andtranslation of the genome; e.g., mRNA, cDNA, or a protein) thereof canbe used. Particularly preferably, mRNA or cDNA is prepared for use.

In the method for detecting the polynucleotide of the present invention,the “step of detecting the presence of the polynucleotide” may bepracticed by detecting the presence of the polynucleotide represented bySEQ ID NO: 4 (genomic sequence comprising the fusion point) or SEQ IDNO:5 (genomic sequence comprising the fusion point) in the genome of thesample obtained from a test subject or detecting the presence of mRNA orcDNA corresponding to the polynucleotide type v1 of the presentinvention (preferably, the EML4-ALK fusion polynucleotide v1) or thepolynucleotide type v2 of the present invention (preferably, theEML4-ALK fusion polynucleotide v2) by preparing a transcription product(e.g., mRNA or cDNA) of genomic DNA extracted from the sample obtainedfrom a test subject.

The genomic DNA extraction can be performed by a method known in the artand can be performed conveniently with a commercially available DNAextraction kit. Examples of the commercially available DNA extractionkit include, but not limited to, G-DEX™ genomic DNA extraction kit(Cosmo Bio Co., Ltd.) and Get pure DNA Kit-Cell, Tissue (Dojindo Co.,Ltd.).

The detection step at the step (1) can be practiced according to a geneanalysis method known in the art (e.g., PCR commonly used as a genedetection method, LCR (Ligase chain reaction), SDA (Strand displacementamplification), NASBA (Nucleic acid sequence-based amplification), ICAN(Isothermal and chimeric primer-initiated amplification of nucleicacids), LAMP (Loop-mediated isothermal amplification), TMA (Gen-Probe'sTMA system), and well known methods such as a microarray). For example,a hybridization technique using, as a probe, a nucleic acid hybridizingto the polynucleotide of the present invention or a gene amplificationtechnique using, as primers, DNAs hybridizing to the polynucleotide ofthe present invention is utilized. Specifically, a nucleic acid, forexample, mRNA, derived from the sample obtained from a test subject isused for measurement. The mRNA level is measured by a gene amplificationreaction method using primers designed to specifically amplify thepolynucleotide sequence of the present invention. The gene amplificationreaction method is not particularly limited. For example, PCR or nucleicacid amplification using RNA polymerase can be utilized. Primers used inthe detection method of the present invention or primers contained inthe kit for detection of the present invention are not particularlylimited as long as they can specifically amplify the polynucleotidesequence of the present invention. These primers are designed on thebasis of the nucleotide sequence of the polynucleotide of the presentinvention. Primer design for a PCR amplification monitoring method canbe achieved by utilizing primer design software Primer Express (PEBiosystems). PCR products with a large size reduce amplificationefficiency. Therefore, it is appropriate that sense and antisenseprimers should be designed to give amplification products of 1 kb orless in size in the amplification of mRNA or cDNA. More specifically, asense primer (5′-primer) and an antisense primer (3′-primer) aredesigned from an EML4-encoding portion (e.g., any portion within theEML4 gene region of the EML4-ALK fusion polynucleotide (particularly,cDNA)) and from an ALK-encoding portion (e.g., any portion within theALK gene region of the EML4-ALK fusion polynucleotide (particularly,cDNA)), respectively. Preferably, primers contained in the kit fordetection of the present invention, more preferably, most suitableprimers contained in the kit for detection of the present invention, areused. Whether the gene of interest (the whole sequence or its specificportion) is amplified or not can be confirmed by a method suitable foreach amplification technique. For example, for PCR, the PCR products canbe subjected to analysis by agarose gel electrophoresis and ethidiumbromide staining to thereby confirm whether an amplification fragmentwith the size of interest is obtained or not. If the amplificationfragment with the size of interest is obtained, then the polynucleotideof the present invention is present in the sample obtained from a testsubject. In this way, the presence of the polynucleotide of the presentinvention can be detected.

Detection using the hybridization technique is performed, for example,by a northern hybridization, dot-blot, or DNA microarray method.Furthermore, a gene amplification technique such as RT-PCR can beutilized. In the RT-PCR method, the presence of the polynucleotide ofthe present invention can be analyzed more quantitatively by using a PCRamplification monitoring (real-time PCR) method (Genome Res., 6 (10),986, 1996) in the gene amplification process. For example, ABI PRISM7900 (PE Biosystems) can be used as the PCR amplification monitoringmethod. The real-time PCR is a method known in the art and can beperformed conveniently by utilizing a commercially available apparatusand a kit for real-time PCR.

The EML4 gene and ALK gene are distantly positioned on the chromosome inopposite orientations. If the EML4-ALK fusion polynucleotide is absent,therefore, RT-PCR using the primers designed for the respective genesdoes not produce PCR products. Specifically, PCR, even if performed byincreased cycles, does not amplify products in normal cells free fromchromosomal inversion between both the genes and in cases of cancer(e.g., lung cancer) free from EML4-ALK inversion. Thus, cells or tissueshaving the polynucleotide of the present invention can be detected withexceeding sensitivity and with few false positives by PCR using theprimer set and using genomic DNA, mRNA, or cDNA as a substrate.

A method for detecting a fusion protein encoded by the polynucleotide ofthe present invention comprises the step of

(2) Detecting the presence of the polypeptide of the present inventionin a sample obtained from a test subject.

Such a detection step can be practiced by preparing a solubilizedsolution derived from a sample obtained from a test subject (e.g., acancer tissue or cell obtained from the test subject) and detecting thepolypeptide of the present invention (particularly, the EML4-ALK fusionpolypeptide v1) contained therein by an immunological measurement orenzyme activity measurement method using anti-EML4 and anti-ALKantibodies in combination. Preferably, a qualitative or quantitativeapproach using a monoclonal or polyclonal antibody specific to thepolypeptide of the present invention (particularly, the EML4-ALK fusionpolypeptide v1) can be used, such as enzyme immunoassay, two-antibodysandwich ELISA, fluoroimmunoassay, radioimmunoassay, or westernblotting.

More preferably, the presence of the polypeptide of the presentinvention can be detected, as shown in Example 9, by subjecting cellextracts from a cell likely to have the polypeptide of the presentinvention to immunoprecipitation with an anti-EML4 antibody andperforming detection using an anti-ALK antibody for the precipitates. Inthe method of Example 9, immunoprecipitation with the anti-ALK antibodyand detection with the anti-EML4 antibody may also be used. After theimmunoprecipitation and detection thus performed, it is preferred tofurther confirm that the protein detected by the detection antibody hasthe size of the polypeptide of the present invention of interest. Evenif cell extracts of tissues or cells free from the polypeptide of thepresent invention are subjected to immunoprecipitation and detectionusing those 2 antibodies, the polypeptide of the present invention isnot detected therein. The antibodies used in this detection may be anyantibody that can specifically bind to a polypeptide sequence from exon1 to exon 20 (preferably, from exon 1 to exon 13) of EML4 or apolypeptide sequence from exon 21 to exon 30 of ALK, and may bemonoclonal or polyclonal antibodies.

If the polynucleotide of the present invention or the polypeptide of thepresent invention (particularly, the EML4-ALK fusion polypeptide v1) isdetected from the sample obtained from a test subject, the test subjectis a target (patient) having cancer that is shown to be positive for thepolynucleotide of the present invention and serves as a target to whichthe pharmaceutical composition of the present specification isapplicable.

The kit for detection of the present invention comprises at least senseand antisense primers designed to specifically amplify thepolynucleotide of the present invention (preferably, the EML4-ALK fusionpolynucleotide). The set of sense and antisense primers arepolynucleotides that function as primers for amplification of thepolynucleotide of the present invention (preferably, the EML4-ALK fusionpolynucleotide). Examples thereof include the primers (1) and (3) to (7)described in the paragraph <Probe or primer of the present invention>.Most preferable primers are the primers (7).

Examples of other reagents that can be contained in the kit fordetection of the present invention can include reagents (e.g., Taqpolymerase, nucleotide substrates, and buffer solutions) necessary forPCR.

<Screening Method of the Present Invention>

The screening method of the present invention encompasses a method forscreening a substance inhibiting the polypeptide of the presentinvention and a method for screening a therapeutic agent for cancer(preferably, lung cancer) that is shown to be positive for thepolynucleotide of the present invention.

(1) Method for screening a substance inhibiting the polypeptide of thepresent invention (inhibiting the activity and/or expression of thepolypeptide of the present invention)

The method for screening a substance inhibiting the polypeptide of thepresent invention (preferably, the EML4-ALK fusion polypeptide v1 or theEML4-ALK fusion polypeptide v2) is not particularly limited as long asit comprises the following steps (i) to (iii):

(i) bringing test substances into contact with the polypeptide of thepresent invention or a cell expressing the polypeptide of the presentinvention;

(ii) analyzing whether the polypeptide is inhibited or not, and

(iii) selecting a substance inhibiting the polypeptide.

If desired, the method for screening a substance inhibiting thepolypeptide of the present invention may further comprise the step ofconfirming that the selected test substance has a therapeutic activityagainst cancer that is shown to be positive for the polynucleotide ofthe present invention.Preferably, the substance inhibiting the polypeptide of the presentinvention can be screened by methods described in Examples 7(3), 7(4),8(5), 8(6), 8(3), 8(7), 8(8), and 8(9).

(2) Method for screening a therapeutic agent for cancer (preferably,lung cancer) that is shown to be positive for the polynucleotide of thepresent invention.

As shown in Examples below (Example 6(1)), a transformed focus, afeature of cancer cells, was formed by causing a 3T3 normal cell toexpress the EML4-ALK fusion polynucleotide v1. When this cell wassubcutaneously inoculated into a nude mouse, tumor was formed, showingthat the EML4-ALK fusion polynucleotide v1 is an oncogene.Alternatively, similar analysis using a variety of mutants (Example6(2)) and analysis of transformability and tumorigenicity (Example10(3)) showed that the EML4-ALK fusion polynucleotide v2 is also anoncogene. Furthermore, the presence of the EML4-ALK fusionpolynucleotide was detected in some lung cancer patients.

Thus, the therapeutic agent for cancer (preferably, lung cancer) that isshown to be positive for the polynucleotide of the present invention canbe screened by selecting a substance inhibiting the polypeptide of thepresent invention. From the novel findings gained by the presentinventors that the anchorage-independent cell growth was inhibited(i.e., an anti-cancer effect is exhibited) by inhibiting the activityand/or expression of the polypeptide of the present invention (Example8), it was shown that the substance inhibiting the polypeptide of thepresent invention (inhibiting the activity and/or expression of thepolypeptide of the present invention) has a therapeutic effect oncancer. Specifically, the method for screening a substance inhibitingthe polypeptide of the present invention (inhibiting the activity and/orexpression of the polypeptide of the present invention) can be utilizedas a method for screening a therapeutic agent for cancer (preferably,lung cancer) that is shown to be positive for the polynucleotide of thepresent invention.

The substance obtained by the screening method (i) to (iii) can besubjected to an evaluation system known in the art about therapeuticagents for cancer or a modified evaluation system thereof to determinewhether or not the substance is useful as a therapeutic agent forcancer. For example, a therapeutic effect on cancer that is shown to bepositive for the polynucleotide of the present invention can beconfirmed and determined on the basis of the inhibitory effect of theinhibiting substance on the anchorage-independent growth of a cellexpressing the polynucleotide of the present invention or on the basisof the inhibitory effect of the inhibiting substance on the growth oftumor formed in a nude mouse inoculated with a cell expressing thepolynucleotide of the present invention. Alternatively, the substanceobtained by the screening method can be subjected to an evaluationsystem similar to that for the therapeutic effect using a lung cancercell expressing the polynucleotide of the present invention to therebydetermine whether or not the substance is useful as a therapeutic agentfor lung cancer that is shown to be positive for the polynucleotide ofthe present invention.

The screening method of the present invention encompasses the followingmethods:

(a) In vitro screening method;

a method for screening a substance inhibiting the activity of thepolypeptide of the present invention, comprising the steps of (1)bringing test substances into contact with the polypeptide of thepresent invention, (2) analyzing whether the activity of the polypeptideis inhibited or not, and (3) selecting a substance inhibiting theactivity of the polypeptide;

(b) Cell-based screening method;

a method for screening a substance inhibiting the activity of thepolypeptide of the present invention, comprising the steps of (1)bringing test substances into contact with a cell expressing thepolypeptide of the present invention, (2) analyzing whether the activityof the polypeptide is inhibited or not, and (3) selecting a substanceinhibiting the activity of the polypeptide; and

(c) Expression inhibition-based screening method;

a method for screening a substance inhibiting the expression of thepolypeptide of the present invention, comprising the steps of (1)bringing test substances into contact with a cell expressing thepolypeptide of the present invention, (2) analyzing whether theexpression of the polypeptide is inhibited or not, and (3) selecting asubstance inhibiting the expression of the polypeptide.

Each screening method will be described below.

<In Vitro Screening Method>

The in vitro screening method comprises: bringing test substances intocontact with the purified polypeptide of the present invention byaddition (contact step); analyzing whether the activity of thepolypeptide of the present invention is inhibited or not by the testsubstance(s), by comparison with the activity of the polypeptide of thepresent invention not brought into contact with the test substances(analysis step); and selecting a substance inhibiting the activity ofthe polypeptide of the present invention (i.e., a therapeutic agent forcancer, particularly, a therapeutic agent for lung cancer).

In the screening method of the present invention, each step canspecifically be practiced, for example, as described below. Thepolypeptide of the present invention is expressed in cells (e.g., BA/F3cells). The expressed polypeptide is isolated and purified from thecells by affinity purification using affinity for a tag such as GST,Flag, or His or by immunoprecipitation using an antibody responding tothe polypeptide of the present invention (e.g., an anti-EML4, anti-ALK,or tag antibody). Subsequently, test substances are brought into contactwith the purified polypeptide by addition. After the addition of ATP,the activity of the polypeptide is measured. Solvents (e.g., DMSO) forthe test substances are brought as a control into contact with thepurified polypeptide by mixing. After the addition of ATP, the activityof the polypeptide is measured. A condition without the addition of ATPcan be set as a background control. Whether the activity (i.e.,phosphorylating activity) of the polypeptide of the present invention isinhibited or not by the test substance(s) is analyzed. Whether theactivity (i.e., phosphorylating activity) of the polypeptide of thepresent invention is inhibited or not by the test substance(s) can bedetermined by analyzing a test substance-induced change in the tyrosinephosphorylation level of the polypeptide of the present invention.Specifically, when the addition (i.e., contact) of a test substanceinhibits the activity (i.e., phosphorylating activity) of thepolypeptide of the present invention as compared with the addition(i.e., contact) of the solvent control, this test substance is selectedas a substance inhibiting the activity of the polypeptide of the presentinvention (i.e., a therapeutic agent for cancer, particularly, atherapeutic agent for lung cancer). Of the screening methods of thepresent invention, preferably, the in vitro screening method ispracticed under the conditions described in Example 7(3). A substancethat can inhibit 50% or more activity by this method at a concentrationof 10 μM or lower, preferably 1 μM or lower, more preferably 0.1 μM orlower is selected as a substance inhibiting the activity of thepolypeptide of the present invention.

<Cell-Based Screening Method>

The cell-based screening method comprises: bringing test substances intocontact with a cell expressing the polypeptide of the present invention(preferably, a cell caused to express the polypeptide of the presentinvention) by mixing (i.e., addition) (contact step); analyzing whetherthe activity of the polypeptide of the present invention is inhibited ornot by the test substance(s), by comparison with the activity of thepolypeptide of the present invention not brought into contact with thetest substances (analysis step); and selecting a substance inhibitingthe activity of the polypeptide of the present invention (i.e., atherapeutic agent for cancer, particularly, a therapeutic agent for lungcancer). This screening method can specifically be practiced, forexample, as described below.

First, test substances or solvent controls (e.g., DMSO) are brought intocontact with a cell expressing the polypeptide of the present invention(i.e., a cell naturally expressing the polypeptide of the presentinvention or a cell caused to express the polypeptide of the presentinvention by its transformation with a vector comprising thepolynucleotide of the present invention). The cells are cultured for agiven time. The activity (i.e., autophosphorylating activity) of thepolypeptide of the present invention is measured using cell lysatesprepared from the cultured cells by dissolution, by SDS electrophoresisknown in the art and immunoblotting using an anti-phosphorylated ALKantibody (e.g., Cell Signaling Technology) to thereby analyze whetherthe activity (i.e., autophosphorylating activity) of the polypeptide ofthe present invention is inhibited or not by the test substance(s).Whether the activity (i.e., autophosphorylating activity) of thepolypeptide of the present invention is inhibited or not by the testsubstance(s) can be determined by analyzing a test substance-inducedchange in the tyrosine phosphorylation (i.e., autophosphorylation) levelof the polypeptide of the present invention. Specifically, when theaddition (i.e., contact) of a test substance inhibits the activity(i.e., autophosphorylating activity) of the polypeptide of the presentinvention as compared with the addition (i.e., contact) of the solventcontrol, this test substance is selected as a substance inhibiting theactivity of the polypeptide of the present invention (i.e., atherapeutic agent for cancer, particularly, a therapeutic agent for lungcancer). Of the screening methods of the present invention, preferably,the cell-based screening method is practiced under the conditionsdescribed in Example 7(4) or 10(2). A substance that can inhibit 50% ormore activity by this method at a concentration of 10 μM or lower,preferably 1 μM or lower, more preferably 0.1 μM or lower is selected.

<Expression Inhibition-Based Screening Method>

The expression inhibition-based screening method comprises: bringingtest substances into contact with a cell expressing the polypeptide ofthe present invention (preferably, a cell caused to express thepolypeptide of the present invention) by mixing (i.e., addition)(contact step); analyzing whether the expression of the polypeptide ofthe present invention is inhibited or not by the test substance(s), bycomparison with the expression of the polypeptide of the presentinvention not brought into contact with the test substances (analysisstep); and selecting a substance inhibiting the expression of thepolypeptide of the present invention (i.e., a therapeutic agent forcancer, particularly, a therapeutic agent for lung cancer, that is shownto be positive for the polynucleotide of the present invention). Thisscreening method can specifically be practiced, for example, asdescribed below.

Test substances or solvent controls (e.g., DMSO) are brought intocontact with any cell expressing the polypeptide of the presentinvention (i.e., a cell naturally expressing the polypeptide of thepresent invention or a cell caused to express the polypeptide of thepresent invention by its transformation with a vector comprising thepolynucleotide of the present invention). After culture, extracts areprepared from the cells and subsequently used to analyze whether theexpression of the polypeptide of the present invention is inhibited ornot by the test substance(s). Whether the expression of the polypeptideof the present invention is inhibited or not can be analyzed byanalyzing whether the mRNA or protein expression of the polypeptide ofthe present invention is inhibited or not. More specifically, the mRNAor protein level of the polypeptide of the present invention present inthe cell extracts is identified by an expression level analysis methodknown in the art, for example, northern blotting, quantitative PCR,immunoblotting, or ELISA. More specifically, the inhibition of the mRNAor protein expression of the polypeptide of the present invention can beanalyzed by a method described in Example 8(5) or 8(6). Whether theexpression of the polypeptide of the present invention is inhibited ornot by the test substance(s) can be determined by analyzing a testsubstance-induced change in the expression level of the polypeptide ofthe present invention. Specifically, when the contact of a testsubstance inhibits the expression level (i.e., mRNA or protein level) ofthe polypeptide of the present invention as compared with the contact ofthe solvent control, this test substance is selected as a substanceinhibiting the expression of the polypeptide of the present invention(i.e., a therapeutic agent for cancer, particularly, a therapeutic agentfor lung cancer). Of the screening methods of the present invention,preferably, the expression inhibition-based screening method ispracticed under the conditions described in Example 8(5) or 8(6). Asubstance that can inhibit 50% or more activity by this method at aconcentration of 10 μM or lower, preferably 1 μM or lower, morepreferably 0.1 μM or lower is selected. Preferably, the selected testsubstance has an inhibitory activity on all cells used. However, a testsubstance having an inhibitory activity on one cell can also beselected.

Preferably, the screening method of the present invention furthercomprises, in addition to analyzing whether the polypeptide of thepresent invention is inhibited or not and selecting a substanceinhibiting the polypeptide of the present invention, the step ofconfirming that the selected test substance has a therapeutic activityagainst cancer (particularly, lung cancer) that is shown to be positivefor the polynucleotide of the present invention.

Examples of the step of confirming that the selected substance has atherapeutic activity against cancer (particularly, lung cancer) that isshown to be positive for the polynucleotide of the present inventioninclude a step of practicing an evaluation method known in the art or amodified method thereof, for example, a method comprising analyzing thetherapeutic activity of the selected substance against cancer(particularly, lung cancer) by treating, with the substance, culturedcells or tumor model animals expressing the polypeptide of the presentinvention (Clinical Oncology, 2nd ed., Cancer and ChemotherapyPublishers, Inc.).

Examples of the cultured cells expressing the polypeptide of the presentinvention include human cancer-derived (particularly, lungcancer-derived) cancer cells endogenously expressing the polypeptide ofthe present invention and cells artificially transformed from normalcells such as NIH3T3 by the expression of the polypeptide of the presentinvention. When the therapeutic activity against cancer that is shown tobe positive for the polynucleotide of the present invention is examinedusing the cancer cells endogenously expressing the polypeptide of thepresent invention, a growth-inhibiting effect or cell death-inducingeffect on the cancer cells expressing the polypeptide of the presentinvention by the test substance can be confirmed by adding the testsubstance selected by the screening method of the present invention to aculture medium of the cancer cells and measuring a cell count or celldeath rate after culture by a standard method. If the selected testsubstance exhibits the growth-inhibiting effect and/or celldeath-inducing effect on the cells, this selected test substance isconfirmed to have a therapeutic activity against cancer (particularly,lung cancer) that is shown to be positive for the polynucleotide of thepresent invention. The test substance may be added to the medium underconditions in which the test substance is added at the start of cultureor during culture once or any number of times without limitations. Aculture period in the presence of the test substance can be setappropriately and is 5 minutes to 2 weeks, preferably 1 hour to 72hours. Any of a variety of cell measurement methods may be used, such astrypan blue staining, Sulforhodamine, MTT, intracellular ATPmeasurement, and thymidine uptake methods, and any of a variety of celldeath measurement methods may be used, such as LDH release measurement,annexin V staining, and caspase activity measurement methods.

When the transformed cells caused to express the polypeptide of thepresent invention are used, the inhibitory effect of the test substanceon the growth of the transformed cells can be examined withanchorage-independent growth, one feature of cancer cells, as an indexto thereby determine a therapeutic activity against cancer. Theanchorage-independent growth refers to, in contrast to adherent normalcells that must adhere to the extracellular matrix (anchorage) for theirsurvival and growth, the general essential property of cancer cellscapable of growing even without such an anchorage. One of most reliablemethods for examining the carcinogenesis of cells is to confirm that thecells can grow without an anchorage. Whether cells transformed fromnormal cells by gene expression exhibit an anchorage-independent growthability can be examined to determine whether the gene is an oncogene. Asdescribed above, the EML4-ALK fusion polynucleotide is an oncogene. Thetransformed cells caused to express the polypeptide of the presentinvention also acquire an anchorage-independent growth ability.Therefore, the therapeutic activity of the test substance against cancerthat is shown to be positive for the polynucleotide of the presentinvention can be examined with this property as an index. Theanchorage-independent growth of the transformed cells caused to expressthe polypeptide of the present invention can be achieved by a method forcell culture in a soft agar medium or a method for cell culture in aplate capable of cell-culturing spheroids (cell aggregates). Measurementmay be performed by the cell measurement methods described above. Thetransformed cell used may be any mammalian cell capable of expressingthe polypeptide of the present invention and anchorage-independentlygrowing. Examples thereof include, but not limited to, mousefibroblast-derived cell line NIH3T3 cells caused to express thepolypeptide of the present invention.

In the method using the tumor model animal, the test substance selectedby the screening method of the present invention is administered to amammalian individual that expresses the polypeptide of the presentinvention and forms tumor. The mammalian species that can be used is anon-human mammal and is, preferably, a mouse, rat, or hamster, morepreferably, a mouse or rat. A cancer-bearing model animal serving as atumor model animal can also be used in which the cancer cellsendogenously expressing the polypeptide of the present invention or thecells transformed by the expression of the polypeptide of the presentinvention are transplanted subcutaneously, intradermally, orintraperitoneally or into each organ (e.g., a nude mouse in which NIH3T3cells caused to express the polypeptide of the present invention aretransplanted). Furthermore, an animal caused to overexpress the EML4-ALKfusion polynucleotide can also be used. The therapeutic activity of thetest substance against cancer that is shown to be positive for thepolynucleotide of the present invention can be confirmed byadministering the test substance by a variety of administration methodssuch as oral, intravenous, subcutaneous, and intraperitonealadministrations and measuring the volume or weight of the tumor of themodel animal. Preferably, the therapeutic activity of the selectedsubstance against cancer that is shown to be positive for thepolynucleotide of the present invention can be confirmed by a methoddescribed in Example 8(8).

Examples of the test substances used in the screening method of thepresent invention can include, but not particularly limited to,commercially available compounds (including peptides), a variety ofcompounds (including peptides) known in the art and registered inchemical files, compound groups obtained by a combinatorial chemistrytechnique (N. Terrett et al., Drug Discov. Today, 4 (1): 41, 1999),microorganism culture supernatants, plant- or marine organism-derivednatural components, animal tissue extracts, double-stranded nucleicacids, antibodies or antibody fragments, and compounds (includingpeptides) chemically or biologically modified from compounds (includingpeptides) selected by the screening method of the present invention.

<Method for Treating Cancer that is shown to be Positive for thePolynucleotide of the Present Invention and Double-Stranded NucleicAcid>

The present invention encompasses a method for treating cancer that isshown to be positive for the polynucleotide of the present invention,comprising administering an effective amount of a substance inhibitingthe polypeptide of the present invention (e.g., a substance [e.g., adouble-stranded nucleic acid (including siRNA), protein (including anantibody or antibody fragment), peptide, or other compounds] obtained bythe screening method of the present invention) to a subject in need oftreatment of cancer that is shown to be positive for the polynucleotideof the present invention. As a substance in the method for treatingcancer that is shown to be positive for the polynucleotide of thepresent invention, the following pharmaceutical composition(hereinafter, referred to as pharmaceutical composition of the presentspecification) can be used.

The active ingredient in the pharmaceutical composition of the presentspecification can be selected by the screening method of the presentinvention. Examples of the substance selected by the screening method ofthe present invention can include compounds A to D and a double-strandednucleic acid described in Examples 7 and 8 below respectively.Alternatively, a compound selected by the screening method of thepresent invention from a low-molecular-weight compound with aninhibitory activity against ALK (ALK inhibitor) known in the art can beused as an active ingredient in the pharmaceutical composition of thepresent specification. The ALK inhibitor can be exemplified by ALKinhibitors described in WO 2005/097765 and WO 2005/016894. Particularly,a compound described in Wan W et al., Blood 107: 1617-1623, 2006 as wellas WHI-P131 (4-(4′-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline) andWHI-P154 (4-[(3′-Bromo-4′-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline)(both, EMD Biosciences; hereinafter, WHI-P154 is referred to as acompound A; Marzec M et al., Lab Invest 85: 1544-1554, 2005) can beused. Alternatively,N-[2-(3-chlorophenyl)ethyl]-2-[({[4-(trifluoromethoxy)phenoxy]acetyl}amino)methyl]-1,3-thiazole-4-carboxamide(WO 2005/097765; hereinafter, referred to as a compound B),5-chloro-N⁴-[2-(isopropylsulfonyl)phenyl]-N²-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4-diamine(WO 2005/016894; hereinafter, referred to as a compound C), or2-[(5-bromo-2-{[2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide(WO 2005/016894; hereinafter, referred to as a compound D) can be usedas an ALK inhibitor.

The double-stranded nucleic acid exemplified as an active ingredient inthe pharmaceutical composition of the present specification comprises adouble-stranded nucleic acid (RNA or DNA) portion and, preferably,3′-terminal overhangs of the sense and antisense strands and inducesRNAi. The RNAi is an evolutionarily conserved phenomenon, which occursvia a double-stranded nucleic acid with 21 to 23 bases produced by RNaseIII endonuclease (Genes Dev. 15, 485-490, 2001). The 3′-terminaloverhangs are respectively any nucleic acid with 1 or 2 bases,preferably 2 bases. The number of bases (21 to 23 bases) described aboveis the number of bases of the sense or antisense strand including itsoverhang. The sense and antisense strands can have the same number ofbases or a different number of bases and, preferably, have the samenumber of bases.

For example, U (uridine), A (adenosine), G (guanosine), or C (cytidine)can be used as ribonucleic acids constituting the 3′-terminal overhangsof the double-stranded nucleic acid. For example, dT (deoxythymidine),dA (deoxyadenosine), dG (deoxyguanosine), or dC (deoxycytidine) can beused as deoxyribonucleic acids constituting the 3′-terminal overhangsthereof.

The double-stranded nucleic acid that can be used as an activeingredient in the pharmaceutical composition of the presentspecification comprises a double-stranded portion designed on the basisof bases at positions 1743 to 1761, 1744 to 1762, 1750 to 1768, 1753 to1771, 1756 to 1774, or 1757 to 1775 in SEQ ID NO: 1 and has aninhibitory activity on the expression of the polypeptide of the presentinvention (hereinafter, referred to as a double-stranded nucleic acid ofthe present invention). Examples of preferable aspects of such adouble-stranded nucleic acid include siRNA-1 to siRNA-6 described inExample 8 (i.e., a double-stranded nucleic acid, one strand of whichconsists of the nucleotide sequence represented by SEQ ID NO: 111 andthe other strand of which consists of the nucleotide sequencerepresented by SEQ ID NO: 112; a double-stranded nucleic acid, onestrand of which consists of the nucleotide sequence represented by SEQID NO: 113 and the other strand of which consists of the nucleotidesequence represented by SEQ ID NO: 114; a double-stranded nucleic acid,one strand of which consists of the nucleotide sequence represented bySEQ ID NO: 115 and the other strand of which consists of the nucleotidesequence represented by SEQ ID NO: 116; a double-stranded nucleic acid,one strand of which consists of the nucleotide sequence represented bySEQ ID NO: 117 and the other strand of which consists of the nucleotidesequence represented by SEQ ID NO: 118; a double-stranded nucleic acid,one strand of which consists of the nucleotide sequence represented bySEQ ID NO: 119 and the other strand of which consists of the nucleotidesequence represented by SEQ ID NO: 120; and a double-stranded nucleicacid, one strand of which consists of the nucleotide sequencerepresented by SEQ ID NO: 121 and the other strand of which consists ofthe nucleotide sequence represented by SEQ ID NO: 122). Thedouble-stranded nucleic acid of the present invention can be produced bystandard methods (e.g., J. Am. Chem. Soc., 120, 11820-11821, 1998; andMethods, 23, 206-217, 2001). Alternatively, a contract manufacturer fordouble-stranded nucleic acids (e.g., RNAi Co., Ltd.) is well known bythose skilled in the art and can be utilized in the production of thedouble-stranded nucleic acid. The target sequences of the siRNA-1 tosiRNA-6 were confirmed by an siRNA sequence design system (commercialversion siDirect (registered trademark), RNAi Co., Ltd.) to be specificto the polypeptide of the present invention.

The double-stranded nucleic acid of the present invention can bedesigned on the basis of DNA nucleotide sequences (bases at positions1743 to 1761, 1744 to 1762, 1750 to 1768, 1753 to 1771, 1756 to 1774, or1757 to 1775 in SEQ ID NO: 1) targeted by the siRNA-1 to siRNA-6. Such adouble-stranded nucleic acid inhibits the polypeptide of the presentinvention, as with the siRNA-1 to siRNA-6. For example, siRNA can bedesigned which has a double-stranded portion consisting of an RNAnucleotide sequence directly converted from the whole target DNAnucleotide sequence. Alternatively, chimeric DNA-RNA double-strandednucleic acid (which comprises both RNA and DNA in the identical strand)can be designed which has an RNA sequence converted from any portion ofthe target DNA nucleotide sequence. Furthermore a hybrid double-strandednucleic acid, one strand of which is DNA and the other strand of whichis RNA can be designed and produced for use. The conversion of the RNAnucleotide sequence from the DNA nucleotide sequence described hereinmeans that dT in the DNA nucleotide sequence is converted to U and otherbases, that is, dA, dG, and dC are converted to A, G, and C,respectively.

The double-stranded nucleic acid of the present invention exhibited aninhibitory effect on the anchorage-independent growth of a cellexpressing the polypeptide of the present invention (Example 8(7)).Therefore, the double-stranded nucleic acid of the present invention canbe utilized in the treatment of cancer that is shown to be positive forthe polynucleotide of the present invention, comprising administering aneffective amount thereof to a subject in need of treatment of cancerthat is shown to be positive for the polynucleotide of the presentinvention.

A therapeutic effect on cancer that is shown to be positive for thepolynucleotide of the present invention can be confirmed by use of amethod generally known by those skilled in the art or a modified methodthereof (see the “step of confirming that the selected substance has atherapeutic activity against cancer).

A preparation comprising, as an active ingredient, a substanceinhibiting the polypeptide of the present invention (e.g., a substance[e.g., a double-stranded nucleic acid, protein (including an antibody orantibody fragment), peptide, or other compounds] obtained by thescreening method of the present invention) can be prepared as apharmaceutical composition using pharmacologically acceptable carriers,excipients, and/or other additives usually used in the preparationproduction according to the type of the active ingredient.

Examples of administration can include: oral administration usingtablets, pills, capsules, granules, subtle granules, powders, or oralliquid agents; and parenteral administration using injections forintravenous injection (including intravenous drip), intramuscularinjections, or subcutaneous injection, suppositories, percutaneousadministration agents, or transmucosal administration agent.Particularly, parenteral administration such as intravenous injection ispreferable for peptides that are digested in the stomach.

To prepare a solid composition for oral administration, 1 or more activesubstances can be mixed with at least one inactive diluent, for example,lactose, mannitol, glucose, microcrystalline cellulose,hydroxypropylcellulose, starch, polyvinyl pyrrolidone, or magnesiumaluminometasilicate. The composition can contain additives other thanthe inactive diluent, for example, lubricants, disintegrants,stabilizers, or solvents or solubilizers according to a standard method.Tablets or pills can be coated, if necessary, with a sugar coating orwith a film such as a gastrosoluble or enteric substance.

A liquid composition for oral administration can comprise, for example,an emulsion, solution, suspension, syrup, or elixir and can contain aninactive diluent generally used, for example, purified water or ethanol.The composition can contain additives other than the inactive diluent,for example, moisturizers, suspensions, sweeteners, flavors, orantiseptics.

A parenteral injection can comprise an aseptic aqueous or non-aqueoussolution, suspension, or emulsion. A water-soluble solution orsuspension can contain, for example, distilled water or saline forinjection, as a diluent. A water-insoluble solution or suspension cancontain, for example, propylene glycol, polyethylene glycol, plant oil(e.g., olive oil), alcohols (e.g., ethanol), or polysorbate 80 as adiluent. The composition can further contain moisturizers, emulsifiers,dispersants, stabilizers, solvents or solubilizers, or antiseptics. Thecomposition can be sterilized, for example, by filtration using abacterium-impermeable filter, formulation of germicides thereinto, orirradiation. Alternatively, an aseptic solid composition can be producedand dissolved in aseptic water or other aseptic media for injection foruse.

A dose can be determined appropriately in consideration of the activityintensity of the active ingredient, that is, the substance obtained bythe screening method of the present invention, conditions, the age orgender of a subject receiving administration, and so on. Preferably, thedose can be calculated according to a route as an amount that gives aserum concentration around tumor or intratumoral concentration 3 to 30timers, for example, 10 times, higher than a drug concentrationinhibiting 50% of the activity or expression of the polypeptide of thepresent invention. For example, the dose in oral administration in adult(60 kg in body weight) is usually approximately 0.1 to 100 mg/day,preferably, 0.1 to 50 mg/day. The dose in parenteral administration is0.01 to 50 mg/day, preferably, 0.01 to 10 mg/day, in terms of aninjection.

A therapeutic target by the pharmaceutical composition of the presentspecification is a test subject from which the presence of thepolynucleotide of the present invention (preferably, the polynucleotidetype v1 of the present invention and/or the polynucleotide type v2 ofthe present invention, particularly preferably, the EML4-ALK fusionpolynucleotide v1 and/or the EML4-ALK fusion polynucleotide v2) and/orthe polypeptide of the present invention (preferably, the polypeptidetype v1 of the present invention and/or the polypeptide type v2 of thepresent invention, particularly preferably, the EML4-ALK fusionpolypeptide v1 and/or the EML4-ALK fusion polypeptide v2) has beendetected. The substance inhibiting the polypeptide of the presentinvention kills cells that have transformed due to the EML4-ALK fusionpolynucleotide v1. Therefore, the substance inhibiting the polypeptideof the present invention serves as an effective therapeutic agent forcancer (particularly, lung cancer) that is shown to be positive for thepolynucleotide of the present invention.

EXAMPLES

The present invention will be described in detail below by Examples, butthe present inventions are not limited by these Examples. Further,unless otherwise stated the process of the present invention can becarried out according to publicly known methods. Also, commerciallyavailable reagents and kits can be used in accordance with theinstructions of the commercial products.

A full length ALK cDNA was kindly supplied by Dr. Steve Morris of St.Jude Children's Research Hospital. Further, this research project wasapproved by the ethics committee for gene analysis study at JichiMedical University.

Anti-phosphorylated ALK antibody and anti-ALK antibody used wereproduced by Cell Signaling Technology Inc. and NEOMARKERS Inc.,respectively.

Example 1 Isolation of EML4-ALK Fusion Polynucleotide v1

(1) Construction of cDNA Library

Using a RNA purification kit (RNeasy Mini Column; Qiagen Inc.), RNA wasextracted from a resected specimen of lung adenocarcinoma of a 62 yearold male who gave informed consent and cDNA was synthesized usingreverse transcriptase (Power Script Reverse Transcriptase) and primers(an oligonucleotide of SEQ ID NO: 42 and CDS primer IIA) (all fromClontech Inc.). After selectively amplifying the full length cDNA bypolymerase chain reaction (PCR) (17 cycles of 98° C. for 10 seconds and68° C. for 6 minutes) using a primer (5′-PCR primer IIA; Clontech Inc.)and a polymerase (primeSTAR HSDNA polymerase, Takara Bio Inc.), a BstX1adapter (Invitrogen Co.) was attached to the both ends of cDNA. The cDNAthus obtained was ligated to a retrovirus plasmid, and a retrovirusplasmid library was constructed by introducing this plasmid to E. coliDH10B (Invitrogen Inc.). As the result, the plasmid library containingclones more than 1,500,000 colony forming units in total has beensuccessfully constructed.

(2) Focus Formation Assay

2 μg of the plasmid of the library described above and 0.5 μg of aplasmid for packaging (pGP, and pE-eco, both of which were obtained fromTakara Bio Inc.) were transfected to BOSC23 packaging cells using atransfection reagent. Two days after the transfection, the culturesupernatant was recovered as a solution of recombinant retroviruslibrary, mixed with polybrene (Sigama Inc.) at a concentration of 4μg/ml, and the mixture was added to mouse 3T3 cells at MOI (multiplicityof infection) of 0.1 concentration. Two days later, the culturesupernatant of 3T3 cells was changed to DMEM-F12 medium (InvitrogenInc.) supplemented with 5% bovine serum (Invitrogen Inc.) and 2 mML-glutamine, and cells were cultured 2 more weeks to obtain 10 or morekinds of transformed foci. After isolating each 3T3 cell clone, theculturing of the clones was continued separately, and the genomic DNA ofeach clone was extracted. The viral cDNA integrated in each 3T3 clonewas amplified and recovered by carrying out PCR (30 cycles of 98° C. for10 seconds and 68° C. for 6 minutes) using 10 ng of the genomic DNA as atemplate, 5′-PCR primer IIA primer and DNA polymerase (PrimeStar HS DNApolynerase; Takara Bio Inc.), and cloned in pT7Blue-2 vector.

One of the cDNA thus obtained was 3926 base pair long (SEQ ID NO: 1) andhad a single long open reading frame (from the 271st to 3447th base ofSEQ ID NO: 1) coding for a protein having 1059 amino acid residues (SEQID NO: 2). Interestingly, about half of the amino-terminus of theprotein (1-496 amino acid residues of SEQ ID NO: 2), coded by thepresent cDNA having a novel full-length sequence, was perfectly matchedto 1-496 amino acid residues of echinoderm microtubule associatedprotein like-4; EML4 (GenBank accession No. NM_(—)019063), and on theother hand, about half of the carboxyl terminus (497-1059 amino acidresidues of SEQ ID NO: 2) was perfectly matched to the amino acidsequence of anaplastic lymphoma kinase; ALK (GenBank accession No.AB209477. Also, in the nucleotide sequence (SEQ ID NO: 1) of the cDNAthat we identified, 99.9% of the 35-1759 base matched the 1-1725 base ofthe reported human EML4 cDNA, and the 1760-3677 base of our cDNA (SEQ IDNO: 1) matched the 3613-5530 base of human ALK cDNA by 99.9%. Althoughrespective sequences are a little different from the reported basesequences, any of these differences do not lead to amino acidreplacement, and therefore it may be possible that they are genesequence polymorphism. From the above results, the present cDNA wasbelieved to be a fused cDNA between a part of EML4 cDNA and a part ofALK cDNA. Further, the obtained cDNA (the cDNA of EML4-ALK fusedpolynucleotide v1) contained a domain of ALK tyrosine kinase.

Example 2 Confirmation of EML4-ALK Fusion Polynucleotide v1

The EML4 gene and ALK gene in human are both mapped in the short arm ofthe second chromosome in opposite directions (head to head direction).For the cDNA of the EML4-ALK fusion polynucleotide found in Example 1 tobe produced, respective genomes are needed to be cut in the introndownstream from the exon 13 of the EML4 gene and the intron upstreamfrom the exon 21 of the Alk gene, and re-ligated with the one gene inthe reverse direction. To prove this directly, PCR (after 94° C. for 1minute, 40 cycles of 98° C. for 20 seconds and 68° C. for 9 minutes) wascarried out using primers having the base sequences of SEQ ID NO: 40 (asequence designed in the sense strand of the 3′ terminus of the exon 13of the EML4 gene) and SEQ ID NO: 36 (a sequence designed in theanti-sense strand of the exon 21 of the ALK gene), templates of thegenomic DNA of a patient (ID 33) and the control genomic DNA (thegenomic DNA of peripheral monocytes of normal healthy female (46, XX))and DNA polymerase (LA Taq polymerase; Takara Bio Inc.).

As the result, as shown in FIG. 1, a PCR product having about 4 kbplength was obtained only by the genomic DNA of the present patient. Thatis, it was confirmed as expected that in the genomic level the EML4 geneand the ALK gene were cut at the introns and re-ligated in the reversedirection. Further, this PCR product of about 4 kbp was cloned into apT7Blue-2 vector (Novagen Inc.) and the total base sequence wasdetermined. It was made clear that the cuts were located approximately3.6 kbp downstream from the exon 13 of the EML4 gene and 297 bp upstreamfrom the exon 21 of the ALK gene. The base sequence is shown in SEQ IDNO: 4. SEQ ID NO: 4 is a genomic sequence including the fusion point ofthe EML4 gene and ALK gene.

Example 3 Screening for EML4-ALK Fusion Polynucleotide in ClinicalSpecimens

(1) Detection of EML4-ALK Fusion Polynucleotide using cDNA

cDNAs were synthesized from 33 cases of clinical specimens (resectedspecimens of non-small cell lung cancer) including the case (ID 33) usedin the present Example 1 and 2 and from peripheral monocytes of one caseof a normal healthy subject (46, XX).

To detect the cDNA of EML4-ALK fusion polynucleotide v1, PCR (50 cyclesof 94° C. for 15 seconds, 60° C. for 30 seconds and 72° C. for 1 minute)was carried out using a quantitative PCR kit (QuantiTect SYBR Green;Qiagen Inc.), the cDNAs as substrates prepared from the clinicalspecimens and the normal healthy subject described above andoligonucleotides of SEQ ID NO: 8 and 9 as primers. Using the samespecimens, PCR amplifications of the glyceraldehyde-3-phosphatedehydrogenase (herein after GAPDH) cDNA was tried as a control. Todetect the GAPDH cDNA, oligonucleotides consisting of the nucleotidesequences represented by SEQ ID NO: 10 and 11 were used as primers.Amplified respective samples were electrophoresed with a size marker DNA(Marker: 50 bp ladder, Invitrogen Inc.). As the result, as shown in theupper part of FIG. 2, in the total 3 cases including ID 33, the cDNA ofthe EML4-ALK fusion polynucleotide v1 was detected. Further, in all thecases analyzed, an amplification of the GAPDH cDNA was confirmed clearly(Lower part of FIG. 2). In addition, the base sequence of the PCRproducts identified in the cases of ID 20 and ID 39 were analyzed andthe result confirmed that the sequence was identical to that of ID 33(the 247 bp including the fusion point of the EML4 gene and the ALKgene. SEQ ID NO: 14). That is, the result of the analyses of the 33cases of non-small cell lung cancer confirmed that the fusion of theEML4 gene and the ALK gene occurs in 9.1% of the cases (3/33 cases).Further, in the cases where the cDNA of the EML4-ALK fusionpolynucleotide v1 was positive, one case (ID 20) was a squamous cellcarcinoma specimen and the other (ID 39) was an adenocarcinoma specimen.

Mutation in the EGFR gene has been known to be one of the causes of lungcancer. In the 33 specimens of the cases analyzed as described above,the analysis of the presence of an abnormality in the base sequence ofthe EGFR gene according to the known method confirmed a partial deletionof the exon 19 in 6 cases. The cases having the EGFR gene mutation andthe cases positive for the EML4-ALK fusion polynucleotide belonged todifferent subgroups. That is, the existing therapeutic agents for lungcancer, which are therapeutic agent for the lung cancer caused by themutation in the EGFR gene, are expected to be not effective for the lungcancer patients who are positive for the EML4-ALK fusion polynucleotide.

Also, the 33 cases analyzed as described above were subjected to theinvestigation whether the full length ALK gene existed, and it was foundthat it existed in 8 cases. The specimens of the 7 cases among these 8cases did not contain the EML4-ALK fusion polynucleotide. (That is, thefull length ALK gene did not exist in the 2 cases among the 3 caseswhere the EML4-ALK fusion polynucleotide was positive).

Further, analyses of 42 cases of other non-small cell lung cancer casesby a similar method as described above gave about 1 kbp PCR products in4.8% of the cases (2/42 cases), which are larger than those detected inthe cases of ID 20 and ID 39 cases. These were cloned in pT7Blue-2vector and the base sequence was analyzed. The results indicated thatthese were not the exon 13 of the EML4 gene but a fusion product of theexon 20 of the EML4 gene and the exon 21 of the ALK gene (SEQ ID NO: 3).That is, in these products the fused ALK gene fragment was the same, butthe point of cleavage in the EML4 was different. The cDNA sequence ofthe fusion gene of the exon 1-20 of the EML4 gene and the exon 21-30 ofthe ALK gene, which contains the fusion point of the EML gene and theALK gene found in SEQ ID NO: 3, is shown in SEQ ID NO: 6, and the aminoacid sequence of the polypeptide coded thereby is shown in SEQ ID NO: 7.In the present description, the gene that codes for the proteinconsisiting of the polypeptide represented by SEQ ID NO: 7 is called theEML4-ALK fusion polynucleotide v2. The plasmid produced here in which apartial fragment of the EML4-ALK fusion polynucleotide v2 is cloned isdesignated as EML4-ALKv2 partial/pT7Blue-2.

(2) Detection of the EML4-ALK Fusion Polynucleotide v1 using Genomic DNA

It turned out that the presence of the EML4-ALK fusion polynucleotidecan be detected in samples obtained from the test subjects by PCR usingthe genomic DNA samples extracted from the clinical specimens(especially the lung tissue) of the test subjects as Example 2. Thus, asshown below, detection of EML4-ALK fusion polynucleotide v1 was triedusing various primers. Using 1 ng of pT7Blue-2 vector as a template, inwhich the PCR product of about 4 kbp produced as described above wascloned and using a pair of oligonucleotides having 16 to 20 bases as asense primer and an antisense primer [total 10 pairs of the primer set(SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO:23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ IDNO: 32, and SEQ ID NO: 33 and SEQ ID NO: 34)], PCR (30 cycles of 94° C.for 15 seconds, 55° C. for 30 seconds and 72° C. for 1 minute) wascarried out by a DNA polymerase (rTaq DNA polymerase; Takara Bio Inc.).The results indicated that a single DNA fragment having an expected size(from about 270 to 380 bp) was amplified in each PCR. It became clearfrom the above results that the detection of the EML4-ALK fusionpolynucleotide v1 is possible by carrying out PCR using the genomic DNAextracted from the clinical specimens as a template and various primersets which are thought to specifically detect the presence of theEML4-ALK fusion polynucleotide v1.

(3) Detection of the EML4-ALK Fusion Polynucleotide v2 using Genomic DNA

An oligonucleotide having the base sequence represented by SEQ ID NO: 35in the EML4 exon 20 and an oligonucleotide having the base sequencerepresented by SEQ ID NO: 36 in the antisense side of the ALK exon 21were designed as a sense primer and antisense primer, respectively.Using these and using the genomic DNA of a patient (ID#KL-3121) as atemplate in which the cDNA of the EML4-ALK fusion polynucleotide v2 wasdetected, PCR (after 94° C. for 1 minute, 40 cycles of 98° C. for 20seconds and 68° C. for 6 minutes) was carried out with a DNA polymerase(LA Taq polymerase; Takara Bio Inc.). As the result, a PCR product ofabout 850 bp was obtained. The PCR product was cloned in pT7Blue-2vector, the base sequence was determined and an 853 bp sequence shown inSEQ ID NO: 5 was obtained. The analyses of the sequence revealed thatthe 35 bp located at the 3′ terminus of the EML4 exon 20 and the 544 bpof the intron sequence downstream from the EML4 exon 20 were linked tothe 233 bp of the intron sequence upstream from the ALK exone 21 and the41 bp located at the 5′ terminus of the ALK exon 21. That is, it hasbecome clear that the cleavage of the genome occurred at a location 544bp downstream from the EML4 exon 20 and at a location 233 bp upstreamfrom the ALK exon 21. Using 1 ng of pT7Blue-2 vector as a template, inwhich the 853 bp PCR product prepared as described above was cloned, andusing a pair of oligonucleotides having 16 to 20 bases as a sense primerand an antisense primer [total 10 pairs of the primer set (SEQ ID NO: 37and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 18, SEQ ID NO: 41 andSEQ ID NO: 20, SEQ ID NO: 43 and SEQ ID NO: 22, SEQ ID NO: 45 and SEQ IDNO: 46, SEQ ID NO: 47 and SEQ ID NO: 26, SEQ ID NO: 49 and SEQ ID NO:28, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54,and SEQ ID NO: 55 and SEQ ID NO: 34)], PCR reaction was carried outunder the same conditions as those in Example 3(2). As the result, asingle DNA fragment having the expected size (from about 270 to 380 bp)was amplified in each PCR. It turned out from the above results that thedetection of the presence of the EML4-ALK fusion polynucleotide v2 ispossible by carrying out PCR using the genomic DNA extracted from theclinical specimens as a template and primer sets which are thought tospecifically detect the EML4-ALK fusion polynucleotide v2.

Example 4 Method for Detecting mRNA of the EML4-ALK FusionPolynucleotide

(1) Construction of EML4-ALK Fusion Polypeptide v1 Expression Vector andCloning of EML4-ALK Fusion Polynucleotide v2

From the clone in which the EML4-ALK fusion polynucleotide v1 was clonedin the positive direction (designated as EML4-ALKv1/pT7Blue-2) theinsert was taken out by digesting with restriction enzymes EcoRI andSalI and subcloned at the EcoRI-SalI sites of pMXS (JBC 275,24945-24952, 2000). This was designated as EML4-ALKv1/pMXS. Also, thecDNA of the coding region of EML4-ALK fusion polynucleotide (from codon3 to the last codon) having the sites at the both ends which arerecognized by restriction enzyme EcoRI was amplified by carrying out PCR(25 cycles of 98° C. for 10 seconds, 68° C. for 5 minutes) usingEML4-ALKv1/pT7Blue-2 plasmid as a template and oligonucleotidesconsisting of the base sequence represented by SEQ ID NO: 59 and SEQ IDNO: 60 as primers and a DNA polymerase (PrimeStar HS DNA polymerase).After digesting with EcoRI, this was inserted at the EcoRI site of anexpression vector pcDNA3, which is modified so that the insert can beexpressed with an FLAG tag added to the N-terminus, to produce anexpression plasmid (FLAG-EML4-ALKv1/pcDNA3) for EML4-ALK fusionpolypeptide v1 having the FLAG tag at the N-terminus (hereinafter,FLAG-EML4-ALKv1). Further, the cDNA of FLAG-EML4-ALKv1 was taken outfrom this vector by digesting with restriction enzymes HindIII and XbaI,and after converting the both ends to blunt, an EcoRI-NotI-BamHI adaptor(Takara Bio Inc.) was ligated to both ends. The product was inserted atthe EcoRI site of an expression vector, by which the inserted cDNA andcell surface antigen CD8 can be expressed at the same time (bicistronicvector pMX-iresCD8; J. Biol. Chem. 2001, vol. 276, p39012-39020), toproduce an expression vector which expresses both FLAG-EML4-ALKv1 andCD8. This was designated as FLAG-EML4-ALKv1/pMX-iresCD8.

EML4-ALK fusion polynucleotide v2 was cloned as follows.

Using a full length polynucleotide of EML4 cloned in pT7Blue-2 accordingto the non-patent document 8 as a template for obtaining apolynucleotide fragment coding for EML4 of the EML4-ALK fusionpolynucleotide v2, and using an oligonucleotide represented by SEQ IDNO: 57, in which an EcoRI cleavage sequence is attached to the5′-terminus of the start codon ATG of the EML4 gene exon 1 and anoligonucleotide represented by SEQ ID NO: 58, the sequence of whichconsists of 10 bases of the 5′ terminus of the antisense sequence of theALK gene exon 21 fused to the 5′ terminus of the antisense sequence tothe 20 bases of 3′ terminus of the EML4 gene exon 20, respectively as asense and an antisense primers, and using a DNA polymerase (Pyrobest DNApolymerase; Takara Bio Inc.), PCR (25 cycles of 94° C. for 20 seconds,60° C. for 30 seconds, 72° C. for 1 minute) was carried out to obtain aPCR product of about 2260 bp. This product was designated as PCR productA.

While, using EML-ALKv1/pTBlue-2 produced in the present Example 4(1) asa template to obtain the ALK polynucleotide fragment of the EML4-ALKfusion polynucleotide v2, and using an oligonucleotide represented bySEQ ID NO: 101, the sequence of which consists of the 10 bases of thesense sequence at the 3′ terminus of the EML4 gene exon 20 fused to the5′ terminus of the 20 bases of the sense sequence at the 5′ terminus ofthe ALK gene exon 21 and an oligonucleotide represented by SEQ ID NO:102, in which the Xba I cleavage sequence is attached to the 5′ terminusof the antisense sequence area containing the stop codon present in theALK gene exon 30 as a sense and an antisense primers, respectively, PCRwas carried out under the same condition as that for obtaining PCRproduct A to obtain a PCR product of about 1700 bp. This was designatedPCR product B.

PCR products A and B described above were mixed, and annealing andextension reactions (3 cycles of 94° C. for 1 minute, 55° C. for 30seconds and 72° C. for 2 minutes and 30 seconds) were carried out usinga DNA polymerase (Pyrobest DNA polymerase; Takara Bio Inc.) to obtain aproduct of about 4000 bp. This product was TA-cloned into pCR2.1-TOPOvector using TOPO TA Cloning kit (Invitrogen Inc.), and the basesequence was analyzed. As shown in SEQ ID NO: 6, the result indicatedthat the EML4-ALK fusion polynucleotide v2 which consisted of 2242 basesfrom the start codon ATG of the EML4 gene to the exon 20 and 1691 basesfrom the ALK gene exon 21 to the stop codon of the exon 30 was obtained.

(2) Method for Detecting mRNA of EML4-ALK Fusion Polynucleotide v1 andv2

Using 1 ng of FLAG-EML4-ALKv1/pMX-iresCD8 described in (1) as thetemplate of EML4-ALK fusion polynucleotide v1 and 1 ng of EML4-ALKv2partial/pT7Blue-2 in Example 3 as the template for EML4-ALK fusionpolynucleotide v2, and using a pair of oligonucleotides having 16 to 20bases for each variant as a sense primer and an antisense primer [total10 pairs of the primer set for EML4-ALK fusion polynucleotide v1; (SEQID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO:65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 andSEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ IDNO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO:78, and SEQ ID NO: 79 and SEQ ID NO: 80) and the primer set for EML4-ALKfusion polynucleotide v2; (SEQ ID NO: 81 and SEQ ID NO: 82, SEQ ID NO:83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, SEQ ID NO: 87 andSEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ ID NO: 91 and SEQ IDNO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO:96, SEQ ID NO: 97 and SEQ ID NO: 98, and SEQ ID NO: 99 and SEQ ID NO:100)], PCR (30 cycles of 94° C. for 15 seconds, 55° C. for 30 secondsand 72° C. for 1 minute) was carried out by a DNA polymerase (rTaq DNApolymerase; Takara Bio Inc.). As the result, in all the primer set, asingle DNA fragment, each having the expected size (about from 260 to350 bp), was amplified. From the above results, it was confirmed thatdetection of the presence of the EML4-ALK fusion polynucleotide v1and/or v2 is possible by carrying out RT-PCR according to the presentExample using mRNA extracted from the samples of test subjects as thetemplate.

The results of Example 3 and 4(2) described above indicated that thepresence of the EML4-ALK fusion polynucleotide v1 and v2 can be detectedby using either cDNA or genomic DNA prepared from the clinical specimensobtained from test subjects. This fact suggests that patients having theEML4-ALK fusion polynucleotide can be selected and that a tailor-madetreatment can be practiced by which the patients to be treated by theadministration of inhibitors of the EML4-ALK fusion polynucleotideand/or polypeptide are selected beforehand and then treated.

Example 5 Detection of EML4-ALK Fusion Polynucleotide v1 in Sputum

(1) Production of Mouse BA/F3 Cells expressing FLAG-EML4-ALKv1

A recombinant retrovirus was produced by a similar method as describedbefore using FLAG-EML4-ALKv1/pMX-iresCD8 and a blank vector(pMX-iresCD8), and mouse lymphatic cell line BA/F3 cells were infectedtherewith. Cells expressing CD8 on the cell surface were simply purifiedusing magnetic beads reagent for cell separation and a purificationcolumn (anti-CD8 monoclonal antibody bound magnetic beads and MiniMACSpurification column, both Miltenyi Biotec Inc.).

(2) Detection of EML4-ALK Fusion Polynucleotide v1 in Sputum

After mixing sputum samples of normal healthy subjects with the BA/F3cells expressing EML4-ALK fusion polynucleotide v1 (hereinafter v1expressing BA/F3 cells) at 0/mL, 10 cells/mL, 100 cells/mL, 1000cells/mL and 10,000 cells/mL, cDNA was synthesized by the standardmethod. The presence of the EML4-ALK fusion polynucleotide v1 in sputumwas examined by carrying out a PCR reaction (50° C. for 2 minutes, 95minutes for 15 minutes and further 40 cycles of the following reaction(94° C. for 15 seconds, 60° C. for 30 seconds and 72° C. for 1 minute))using the cDNA described above as a substrate, and oligonucleotidesconsisting of the base sequence represented by SEQ ID NO: 8 and SEQ IDNO: 9 as primers and a quantitative PCR kit (QuantiTect SYBR Green;Qiagen Inc.), and PCR products were obtained. As the results, in everycase except at 0/mL, the presence of EML4-ALK fusion polynucleotidecould be confirmed.

Conventionally cytopathological examination using sputum samples hasbeen an important diagnostic method for lung cancer diagnosis. Thisdiagnosis for lung cancer is based on the presence of atypical cells insputum but reliable diagnosis cannot be made unless many lung cancercells exist in the sputum. However, most of the time, such cases werealready in the advanced stage and it has been almost impossible topractice effective early diagnosis for lung cancer. According to thepresent invention, if the EML4-ALK fusion polynucleotide is present insputum, it became clear that it can be detected by PCR even if a minuteamount.

Example 6 Investigation of Transformability and Tumorgenicity ofEML4-ALK Fusion Polypeptide v1

(1) Analysis for the EML4-ALK Fusion Polypeptide v1

EML4-ALK (K589M)/pMXS, in which the 589th amino acid (ATP binding site),a lysine residue, of the EML4-ALK fusion polypeptide v1 was replacedwith methionine was produced using EML4-ALKv1/pMXS as a substrate andusing a mutation introducing kit (QuickChange Site-Directed MutagenesisKit; Stratagene Inc.). In the reaction, oligonucleotides of SEQ ID NO:103 and SEQ ID NO: 104 were used. The ALK cDNA (Morris, S W et al,Science. 1994 Mar. 4; 263 (5151):1281-4) was cloned to a retrovirusvector pMXS and pMX-iresCD8 according to the standard method (designatedas ALK/pMXS and ALK/pMX-iresCD8, respectively).

EML4-ALKv1/pMXS described above, full length ALK/pMXS, a plasmidexpressing EML4-ALK (K589M)/pMXS and a blank vector without insertedcDNA (pMXS) were transfected to 3T3 fibroblast cells by the phosphatecalcium method and cultured for 21 days. As shown in the upper part ofFIG. 3, many transformation foci were observed only when the EML4-ALKfusion polypeptide v1 expressing vector was transfected. The scale barindicates 100 μm. Further, the same transfected 3T3 cells wereinoculated subcutaneously to nude mice at 5×10⁵ cells/mouse and observedfor 20 days. It turned out also that tumor was formed only when EML4-ALKfusion polypeptide v1 expressing cells were inoculated. The tumorformation numbers (the number of inoculation sites of 3T3 cells and thenumber of tumor formation among them) are as follows. The tumorformation number of the full length ALK expression was 0 among 8, whilethe tumor formation number in the EML4-ALK fusion polypeptide v1expressing cells was 8 among 8. In addition the tumor formation numberof EML4-ALK (K589M) expressing cells was 0 among 8. These resultsdemonstrate that since the full length ALK polypeptide expression doesnot induce tumor but the EML4-ALK fusion polypeptide v1 is tumorgenic,the EML4-ALK fusion polynucleotide v1 is the causal gene of cancer.Also, since the tumorgenicity of EML4-ALK was not observed in EML4-ALK(K589M), it would appear that the tumorgenicity was dependent on thekinase activity. Hereinafter, the 3T3 cells in which EML4-ALK fusionpolypeptide v1 is expressed are designated as the v1 expressing 3T3cells.

(2) Analysis of Various Deletion Mutants of EML4-ALK FusionPolynucleotide v1

Various deletion mutants (ΔBasic deletion mutant, ΔHELP deletion mutant,ΔWD deletion mutant) of the EML4 part of the EML4-ALK fusionpolynucleotide v1 was prepared by PCR reaction usingFLAG-EML4-ALKv1/pMX-iresCD8 as a template and using a cloning kit(ExSite PCR-based Site-Directed Mutagenesis; Stratagene Inc.). Forpreparing ΔBasic deletion mutant (31-140th amino acids of the EML4-ALKfusion polypeptide v1 were deleted), the oligonucleotides having thebase sequences represented by SEQ ID NO: 105 and SEQ ID NO: 106 wereused as the primer set; for preparing ΔHELP deletion mutant (220-296thamino acids of the EML4-ALK fusion polypeptide v1 were deleted) theoligonucleotides having the base sequences represented by SEQ ID NO: 107and SEQ ID NO: 108 were used; and for preparing ΔWD deletion mutant(305-475th amino acids of the EML4-ALK fusion polypeptide v1 weredeleted) the oligonucleotides having the base sequences represented bySEQ ID NO: 109 and SEQ ID NO: 110 were used. Using these deletion mutantplasmids, retrovirus solutions were prepared using a similar method tothat of Example 1 to obtain infected 3T3 cells. These respectiveinfected cells were inoculated subcutaneously to nude mice toinvestigate the tumorgenicity, and it was found that tumors were formedby 3T3 cells expressing ΔHELP deletion mutant and ΔWD deletion mutant.The tumor forming number of respective 3T3 cells expressing ΔBasicdeletion mutant, ΔHELP deletion mutant and ΔWD deletion mutant was 0among 8, 7 among 8 and 8 among 8, respectively. Since no tumor formationwas observed in the ΔBasic deletion mutant, it is demonstrated that the31-140th amino acids of the EML4-ALK fusion polypeptide v1 are importantin tumorgenesis. Since the EML4-ALK fusion polypeptide v2, like theEML4-ALK fusion polypeptide v1, appears to contain the aforementionedthe 31-140th amino acids and the ALK kinase region, the EML4-ALK fusionpolynucleotide v2, like the EML4-ALK fusion polynucleotide, isconsidered to be the causal gene of cancer which codes for thepolypeptide having the transformability and tumorgenicity to 3T3 cells.

Example 7 Method for Screening for the EML4-ALK Fusion PolypeptideInhibitors

(1) Preparation the EML4-ALK Fusion Polypeptide v1

The v1 expressing BA/F3 cells (Example 5(1)) were cultured in RPM1640medium containing 10% of fetal bovine serum to obtain 2.7×10⁹ cells.After washing 3 times with PBS, cells were lysed in a lysis solution (50mM Tris.HCl (pH7.4), 150 mM NaCl, 1% Triton X100, 5 mM EDTA, 5 mM EGTA,1 mM NaVO₄, 1 mM DTT and protease inhibitor cocktail complete). TheEML4-ALK fusion polypeptide v1 present in the supernatant obtained aftera centrifugation was purified using ANTI-FLAG M2 Affinity Gel(SIGMA-ALDRICH Inc) according to the method described in the productinformation document. For washing and elution, the washing solution (50mM Tris.HCl (pH7.4), 250 mM NaCl, 0.05% Brij35, 1 mM EDTA, 1 mM EGTA, 1mM NaVO₄, 1 mM DTT, complete) and the elution solution (50 mM Tris.HCl(pH7.4), 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 0.5 mg/mL FLAG peptide)were used, respectively. Immunoblotting using anti-ALK antibody and antiFLAG M2 antibody (SIGMA-ALDRICH Inc.) and silver staining were carriedout for the eluate to detect the EML4-ALK fusion polypeptide v1. It wasdemonstrated that the EML4-ALK fusion polypeptide v1 can be prepared bythis method.

(2) Detection of the In Vitro Kinase Activity of the EML4-ALK FusionPolypeptide v1

The EML4-ALK fusion polypeptide v1 purified as described above wasdiluted in a reaction solution (15 mM Tris HCl (pH7.4), 0.25 mM MgCl₂,0.01% Tween-20, 2 mM DTT), and then ATP was not added or ATP 20 μM wasadded. The respective mixtures were reacted at room temperature for 1hour. After the reaction, the auto phosphorylated EML4-ALK fusionpolypeptide v1 and the EML4-ALK fusion polypeptide v1 were detected byimmunoblotting using anti-phosphorylated ALK antibody, which recognizesspecifically the product phosphorylated at the 1604th tyrosine residueof ALK, and anti-ALK antibody, and quantitated by an image analysissystem (VersaDoc Imaging System; Bio-Rad Inc.). The amount ofphosphorylation was calibrated by dividing the count of theautophosphorylated EML4-ALK fusion polypeptide v1 by the count of theEML4-ALK fusion polypeptide v1. As the result, the autophosphorylatedEML4-ALK fusion polypeptide v1 band was detected at the location ofabout 130 kDa under the condition of ATP addition, and the amount ofphosphorylation was increased by about 205 folds compared to no-ATPaddition.

In addition, the phosphorylation activity toward a peptide substrate wasinvestigated using a kinase activity detection kit (HTRF KinEASE-TK;Cisbio Inc.). Using TK substrate 1, which was included in the kit, asthe substrate, and after adding no ATP or 100 μM ATP, the mixtures werereacted at room temperature for 1 hour, and the count of HTRF wasdetected as recommended by the Kits manufacturer. As the result itbecame clear that the count of HTRF (that is, phosphorylation of thepeptide substrate) was increased by about 12 times by the addition ofATP compare to no addition of ATP. As shown above, the in vitro kinaseactivity of the EML4-ALK fusion polypeptide v1 can be detected usinganti-phosphorylated ALK antibody and the kinase activity detection kit.

(3) Inhibitory Effect of Compounds against the In Vitro Kinase Activityof the EML4-ALK Fusion Polypeptide v1

The inhibitory effect of compound A-D against the in vitro kinaseactivity of the EML4-ALK fusion polypeptide v1 was investigated usinganti-phosphorylated ALK antibody and the kinase activity detection kit.Respective compounds were added to the reaction solution containing theEML4-ALK fusion polypeptide v1 at a final concentration of 10 μM or 10nM, and then the reaction was carried out with or without the additionof ATP. The rest of the operations were carried out according to themethod (2) described above. In the absence of the compound, thephosphorylation count without ATP addition and with ATP addition wasassumed to be 100% inhibition and 0% inhibition, respectively. Theinhibition (%) of the kinase activity of the EML4-ALK fusion polypeptidev1 by a compound was calculated by the following formula.[Kinase activity inhibition (%) by a compound]=(1-[phosphorylation countwhen the compound and ATP were added−phosphorylation count when thecompound was not added and ATP was not added]/[phosphorylation countwhen the compound was not added and ATP was added−phosphorylation countwhen the compound was not added and ATP was not added])×100

As the result, it was found that compound A-D inhibited thephosphorylation activity of the purified EML4-ALK fusion polypeptide v1on itself and the peptide substrate (Table 1). Compound A and B could beselected as substances which inhibited the activity of the EML4-ALKfusion polypeptide v1 by 50% or more at a concentration of 10 μM orless, and compound C and D could be selected as substances whichinhibited the activity of the EML4-ALK fusion polypeptide v1 by 50% ormore at a concentration of 0.1 μM or less.

TABLE 1 Final Inhibition on Inhibition on Compound concentrationautophosphorylation peptide substrate A 10 μM 104% 99% B 10 μM  68% 56%C 10 nM 102% 99% D 10 nM  96% 99%

The above results indicated that screening (an in vitro type screening)for a substance which inhibits the activity of the polypeptide of thepresent invention could be performed by preparing the EML4-ALK fusionpolypeptide and using the in vitro kinase activity as an index.

(4) Inhibitory Effect of EML4-ALK Fusion Polypeptide v1 Inhibitors onIntracellular Autophosphorylation

(4-1) BA/F3 Cells

Compound A (1 μM, 5 μM and 10 μM) was added to the culture medium of v1expressing BA/F3 cells (Example 5(1)) or not added, and cultured for 3hours. In addition, BA/F3 cells expressing FLAG-EML4-ALKv1(K589M) wereproduced using pMX-iresCD8 vector in which EML4-ALK (K589M) wasintegrated so that FLAG can be added, and cultured. After culturing,cells were counted and the level of phosphorylation of tyrosine ofEML4-ALK fusion polypeptide v1 was measured by immunoblotting usinganti-phosphorylated ALK antibody. Further, for the same transfermembrane, immunoblotting analysis by anti-FLAG tag antibody (EastmanKodak Inc.) was carried out, and total protein quantity of the FLAGattached EML4-ALK fusion polypeptide v1 was measured. As shown in theupper part of FIG. 4, tyrosine phosphorylation level of the EML4-ALKfusion polypeptide v1 was detected in v1 expressing BA/F3 cells, but notyrosine phosphorylation was detected when EML4-ALK (K589M) wasexpressed. This fact indicates that the tyrosine phosphorylation of theEML4-ALK fusion polypeptide v1 detected in BA/F3 cells is anautophosphorylation by the EML4-ALK fusion polypeptide v1 itself Also,it has been confirmed that compound A inhibits the intracellularautophosphorylation of the EML4-ALK fusion polypeptide v1 in aconcentration-dependent manner. In addition, it has been shown that theamount of protein expression itself of the EML4-ALK fusion polypeptidev1 in all the samples has been almost constant (FIG. 4, lower part).

Inhibitory effect of compound B-D on the intracellularautophosphorylation was examined in a similar manner as described above.However, the culturing time after the addition of the compounds was 6hours, and the total protein amount of the EML4-ALK fusion polypeptidev1 was measured using anti-ALK antibody. In addition, the amount ofphosphorylation was calculated by quantitating as in Example 7(2). Also,for compound A, the amount of phosphorylation was calculated byquantitating in experiment under this condition. The rate of inhibitionwas calculated from the amount of phosphorylation when the compound wasadded, using the value when the compound was not added (the solvent ofthe compound, DMSO was added) as a control (0% inhibition). Everycompound clearly inhibited the kinase activity of the EML4-ALK fusionpolypeptide v1 in BA/F3 cells (Table 2). Compound A and B can beselected as the substance which inhibit the EML4-ALK fusion polypeptidev1 activity by 50% or more at a concentration of 10 μM or less, andcompound C and D can be selected as the substance which inhibit theEML4-ALK fusion polypeptide v1 activity by 50% or more at aconcentration of 0.1 μM or less, and it has been demonstrated thatsubstances which inhibit the activity of the polypeptide of the presentinvention can be screened (cell type screening).

(4-2) 3T3 Cells

In a similar manner as in (4-1) except compound A-D were added to v1expressing 3T3 cells (Example 6(i)) at 10 μM or 10 nM and cultured for 4hours, the amount of phosphorylation of tyrosine of EML4-ALK fusionpolypeptide v1 and the total protein amount of the EML4-ALK fusionpolypeptide v1 were measured, and the inhibition rate of respectivecompounds on intracellular kinase activity was calculated. Each compoundinhibited clearly the kinase activity of the EML4-ALK fusion polypeptidev1 in the v1 expressing 3T3 cells (Table 2). It became clear thatvarious cells such as BA/F3 cells, 3T3 cells and the like can be used ascells expressing the polypeptide of the present invention in the celltype screening method of the present invention.

TABLE 2 Inhibition on Inhibition on Final autophosphorylationautophosphorylation Compound concentration (BA/F3cells) (3T3cells) A 10μM 74% 82% B 10 μM 77% 49% C 10 nM 84% 77% D 10 nM 90% 86%

The above results indicated that the kinase activity inhibiting compoundof the EML4-ALK fusion polypeptide v1 activity can be obtained by usingas an index the autophosphorylation in the cells expressing the EML4-ALKfusion polypeptide v1.

Example 8 Growth Inhibitory Effect of the Inhibitors of EML4-ALK FusionPolypeptide on Cells Expressing the EML4-ALK Fusion Polynucleotide v1

(1) Growth Potential of v1 Expressing BA/F3

For growth of BA/F3 cells expressing only CD8 protein (Example 5(1)),BA/F3 cells expressing CD8 as well as ALK, the EML4-ALK fusionpolypeptide v1 (Example 5(1)) or the EML4-ALK (K589M) defecting kinaseactivity, the change in the number of cells starting from 8×10⁵ cells inthe time course was counted in the presence or absence of a growthfactor IL-3. The results are shown in FIG. 5. The v1 expressing BA/F3cells can grow with or without IL-3. However BA/F3 cells expressing onlyCD8 did grow in the presence of IL-3 but died rapidly when IL-3 wasremoved. This fact indicates that the EML4-ALK fusion polynucleotide v1has an activity as oncogene. Further, cells expressing the full lengthhuman ALK and BA/F3 cells expressing EML4-ALK (K589M) that defect kinaseactivity similarly died in the absence of IL-3. These results indicatethat cells obtain the growth potential, even in the absence of thegrowth factor, by expressing the EML4-ALK fusion polypeptide v1 and thatthe growth potential is dependent on the kinase activity of the EML4-ALKfusion polypeptide v1. BA/F3 cells expressing the full length ALK wasobtained according to Example 5(1) and Example 6(1).

(2) Growth Inhibitory Effect of the Inhibitors of the EML4-ALK FusionPolypeptide on v1 Expressing BA/F3 cells

Next, compound A, which is a substance that inhibits the EML4-ALK fusionpolypeptide v1, was added to BA/F3 cells which obtained the IL-3independent growth potential by expressing the EML4-ALK fusionpolypeptide v1, and its effect on cell growth was investigated. Whencompound A was added at 1 μM, 5 μM or 10 μM, or not added (0 μM) to thecontrol, CD8 expressing BA/F3 cells, which grow in the presence of IL-3and the cell growth was measured, cells could grow although a slightgrowth inhibition was observed as shown in FIG. 6( a). On the otherhand, when compound A was added to v1 expressing BA/F3 cells which weregrowing in the absence of IL-3, the cell growth was markedly inhibitedby concentration-dependence of compound A and cell death was induced asshown in FIG. 6( b). That is, it was confirmed that cells, growingdependently on the EML4-ALK fusion polynucleotide (oncogene), werekilled by an inhibitor of the EML4-ALK fusion polypeptide v1.

(3) Inhibitory Effect of the Inhibitors of EML4-ALK Fusion Polypeptideon Anchorage Independent Growth of Cells Expressing the EML4-ALK FusionPolypeptide v1 and the Full Length ALK Polypeptide

Measurement for anchorage independent cell growth (colony method, etc)has been known to be a system for investigating an antitumor effect(pharmacologic effect) of compounds (Clinical Oncology, second edition,Cancer and Chemotherapy Publishers Inc.). In place of the colony method,there is a following method using spheroid plates for measuring thegrowth of non-attached cells.

The v1 expressing 3T3 cells (Example 6(1)) and one of the human gliomacells expressing the full length ALK polypeptide and not expressingEML4-ALK fusion polynucleotide endogenously, U-87 MG cells, were seededto a 96 well spheroid plate (Sumilon Celltight Spheroid 96U, SumitomoBakelite Inc.) at 3000 cells per well in a medium containing 10% fetalbovine serum (DMEM for v1 expressing 3T3 cells and RPMI 1640 forU-87MG). Under 5% CO₂, cells were cultured overnight at 37° C., and thencompound A or B was added to a final concentration of 10 μM, compound Cor D was added to a final concentration of 10 nM and as a negativecontrol the solvent of the compounds, DMSO, was added to make the sameconcentration as the compounds. At the same time, cells were countedbefore the addition of drugs (Day 0). Then, cells were cultured under 5%CO₂, at 37° C. for 3 days, mixed with a reagent for measuring cellnumber (CellTiter-Glo™ Luminescent Cell Viability Assay; Promega Inc.)stirred for 20 minutes, and the measurements (day 3) were carried outusing a luminometer (ML3000 microtiter plate luminometer; DynatechLaboratories Inc.). The results show that all the compounds had growthinhibitory activity on v1 expressing 3T3 cells but almost no inhibitoryactivity on U-87MG cells. The inhibition rate of the compounds wascalculated assuming the cell number at Day 0 and Day 3 were 100%inhibition and 0% inhibition, respectively (Table 3).

TABLE 3 Final v1 expressing U-87MG Compound concentration 3T3 cells cellA 10 μM 106% 15% B 10 μM  91% 34% C 10 nM 131% −2% D 10 nM 135% −2%

Above results indicate that compound A-D inhibited the anchorageindependent cell growth of v1 expressing 3T3 cells by inhibiting thekinase activity of the EML4-ALK fusion polypeptide v1. In addition, itbecame clear that these compounds could not inhibit the anchorageindependent cell growth of U-87MG cells expressing the full length ALKpolypeptide. These results indicate that the inhibitors of EML4-ALKfusion polypeptide can inhibit the growth of cancer cells and tumorswhich express the EML4-ALK fusion polypeptide.

(4) Preparation of siRNA to the EML4-ALK Fusion Polynucleotide v1

siRNAs, which were composed of a sense strand consisting of the basesequences represented by SEQ ID NO: 111, 113, 115, 117, 119, or 121, andan antisense strand consisting of the base sequences represented by SEQID NO: 112, 114, 116, 118, 120, or 122, were prepared as the siRNA(siRNA-1 to siRNA-6) which have a 100% homology to the fusion area ofthe EML4-ALK fusion polypeptide v1 and is expected to have inhibitoryactivity on the expression of EML4-ALK fusion polypeptide v1. Inaddition, siRNAs composed of a sense strand consisting of the basesequences represented by SEQ ID NO: 123 or 125 and an antisense strandconsisting of the base sequence represented by SEQ ID NO: 124 or 126were designed and prepared, as the siRNA (siRNA-7, siRNA-8) which shows100% homology to the ALK area of the EML4-ALK fusion polypeptide v1 andare expected to have an inhibitory effect on the expression of the ALKgene, using an siRNA sequence design system (Commercial siDirect(registered trade mark) RNAi Inc.). It has been confirmed by the siRNAsequence design system (Commercial siDirect (registered trade mark) RNAiInc.) that the base sequences corresponding to siRNA-1 to siRN-8 do notshow a 100% homology to the gene other than the EML4-ALK fusionpolynucleotide and ALK genes. For control experiments to investigate theinfluence of non specific siRNA, siRNA composed of a sense strandconsisting of a base sequence represented by SEQ ID NO: 127 and anantisense strand consisting of a base sequence represented by SEQ ID NO:128 was prepared as siRNA (siRNA-9) corresponding to a base sequence notpresent in mammalian cells. siRNA-1 is a product of annealing SEQ ID NO:111 (sense strand) and SEQ ID NO: 112 (antisense strand), and siRNA-2and others are the same (FIG. 7).

(5) Inhibitory Effect of siRNA on mRNA Expression of the EML4-ALK FusionPolynucleotide and ALK Gene in Cells Expressing the EML4-ALK FusionPolynucleotide v1 and Full Length ALK

The v1 expressing 3T3 cells and U-87MG cells were seeded to 12 wellplates (IWAKI; Asahi techno glass corp.) at 50,000 cells and 150,000cells, respectively. Four hours later, using a transfection reagent(Lipofectamine RNAiMax; Invitrogen Inc.), siRNA-1 to siRNA-9 wereprepared to a final concentration of 20 nM and transfected to cellsaccording to the attached instruction. In addition, as a control nosiRNA transfection samples were prepared. After 72 hours, the medium wasremoved, and total RNA was extracted using an RNA purification kit(RNeasy Mini Kit; QIAGEN Inc.) according to the attached instruction,and cDNA was prepared using a cDNA synthesizing kit (ThermoScript RT-PCRSystem; Invitrogen Inc.) according to the attached instruction.

The amount of expressed mRNA of the EML4-ALK fusion polynucleotide v1 inv1 expressing 3T3 cells and the amount of mRNA of the ALK gene in U-87MGcells were quantitated using a quantitative PCR reagent (Power SYBRGreen PCR Master Mix; Applied Biosystems Inc.) The PCR reaction wascarried out as follows: after 10 minutes incubation at 95° C., 45 cyclesof 95° C. for 15 seconds and 60° C. for 60 seconds, and then one cycleof 95° C. for 15 seconds, 60° C. for 15 seconds and 95° C. for 15seconds to complete the reaction. In addition, to calibrate the amountof expression, the amount of expression of the mouse cyclophilin B geneand the human GAPDH gene was similarly quantitated for v1 expressing 3T3cells and U-87MG cells, respectively. Analyses were carried out using asequence detector (ABI PRISM 7900 Sequence Detection System;Perkin-Elmer Applied Biosystems Inc.).

Oligo nucleotides consisting of the base sequences represented by SEQ IDNO: 44 and 48, SEQ ID NO: 50 and 56, SEQ ID NO: 44 and 48 and SEQ ID NO:12 and 13 were used as the primer sets that specifically recognize theEML4-ALK fusion polynucleotide v1, the mouse cyclophilin B gene, thehuman ALK gene and the human GAPDH gene. Also, to obtain a standardcurve to calculate the amount of respective mRNA, PCR was performedusing human genomic DNA (Clontech) as a template for EML4-ALK fusionpolynucleotide v1, human ALK and human GAPDH, and using mouse genomicDNA (Clontech) as a template for mouse cyclophilin B and theaforementioned primer sets under the same condition. Since a primer setcorresponding to human ALK polynucleotide exon 29 was used to detect theEML4-ALK fusion polynucleotide v1, the standard curve can be obtainedusing human genomic DNA. The expression amount of the EML4-ALK fusionpolynucleotide v1 and the human ALK gene in respective samples wascalibrated with the expression amount of the mouse cyclophilin B geneand the human GAPDH gene to obtain the calibrated expression amount.Further, assuming the respective calibrated expression amount of theEML4-ALK fusion polynucleotide v1 and the human ALK gene in the absenceof siRNA to be 100%, the relative expression amount of the calibratedexpression amount of the EML4-ALK fusion polynucleotide v1 and human ALKgene was determined and the inhibition rate for expression wascalculated when respective siRNAs were transfected (Table 4).

As the result, siRNA-1 to siRNA-8, which correspond to the EML4-ALKfusion polynucleotide v1, inhibited the expression of the EML4-ALKfusion polynucleotide by 50% or more. For the human ALK gene, siRNA-7and siRNA-8, which correspond to the human ALK gene, inhibited by 50% ormore. The negative control, siRNA-9, did not demonstrate strongexpression inhibitory effect on the EML4-ALK fusion polynucleotide andthe human ALK gene. These results demonstrate that substances inhibitingthe expression of the EML4-ALK fusion polynucleotide can be screened.

TABLE 4 Inhibition rate for Inhibition rate for EML4-ALK gene expressionALK gene expression siRNA (v1 expressing 3T3 cells) (U-87 MG cells)siRNA-1 80% −12% siRNA-2 66% 11% siRNA-3 62% 10% siRNA-4 86% 22% siRNA-576% −22% siRNA-6 69% 15% siRNA-7 67% 66% siRNA-8 70% 58% siRNA-9 29%−31% siRNA not  0% 0% introduced(6) Inhibitory Effect of siRNA on Expression and Autophosphorylation ofthe EML4-ALK Fusion Polypeptide v1 in v1 Expressing 3T3 Cells.

By the similar method described before, v1 expressing 3T3 cells wereseeded to 12 well plates at 50,000 cells per well, and 4 hours later,siRNA-1 to siRNA-9 were transfected. In addition, cells to which nosiRNA was transfected were prepared as a control. After 72 hours of thetransfection, the medium was removed, and the autophosphorylation of theintracellular EML4-ALK fusion polypeptide v1 and protein amount of theEML4-ALK fusion polypeptide v1 were quantitated by the similar method asin Example 7(4). Also, to confirm that total protein in each sample formeasurement was the same amount, the actin protein was quantitated usinganti-actin antibody (SIGMA-ALDRICH Inc.). siRNA-1 to siRNA-8 wereclearly inhibited the expression of the EML4-ALK fusion polypeptide v1and the kinase activity in the v1 expressing 3T3 cells.

(7) Growth Inhibitory Effect of siRNA on v1 Expressing 3T3 Cells andFull Length ALK Expressing Cells

Cell growth inhibition rate was calculated with or without transfectingsiRNA by the similar method as in Example 8(3), except that each siRNAwas added to 96 well spheroid plates beforehand and then v1 expressing3T3 cells and U-87MG cells were seeded and cultured for 3 days.

Results of these experiments are shown in Table 5. Each siRNA (siRNA-1to siRNA-8), which had been demonstrated to have inhibitory effect onthe expression of the EML4-ALK fusion polypeptide v1 in Example 8(5)(6), strongly inhibited anchorage independent growth of v1 expressing3T3 cells. Since the cell number of v1 expressing 3T3 cells, to whichsiRNA-1, 3, 4 and 5 were transfected, was lower at the measuring time(Day 3) than when siRNA was transfected (Day 0) resulting in the growthinhibition rate being over 100%, cell death is believed to have beeninduced. On the other hand, siRNA-7 and siRNA-8 were shown to inhibitthe expression of the ALK gene in Example 8(5) but did not inhibit thegrowth of U-87MG cells expressing the full length ALK. siRNA-1 tosiRNA-6 did not show growth inhibition on U-87MG cells and the negativecontrol, siRNA-9, did not inhibit the growth of v1 expressing 3T3 cellsand U-87MG cells.

TABLE 5 v1 expressing 3T3 cells U-87MG cell growth siRNA growthinhibition rate inhibition rate siRNA-1 110% −13% siRNA-2  94% 25%siRNA-3 108% −21% siRNA-4 120% −8% siRNA-5 110% −13% siRNA-6  97% 16%siRNA-7  87% 15% siRNA-8  94% −21% siRNA-9 −35% −16% siRNA notintroduced  0% 0%

From the above results, it became clear that, by transfecting siRNAwhich is against the EML4-ALK fusion polynucleotide v1 to cancer cellsexpressing the EML4-ALK fusion polynucleotide v1, the expression of theEML4-ALK fusion polynucleotide v1 mRNA is inhibited resulting in thereduction of the EML4-ALK fusion polypeptide and in the inhibition ofautophosphorylation causing the growth inhibition of cancer cells. Also,for cancer cells expressing the full length ALK, the growth inhibitiondid not occur when the expression of ALK was inhibited. From the aboveresults, it became clear that the siRNA against the EML4-ALK fusionpolynucleotide v1, for example, siRNA-1 to siRNA-8, is useful as atherapeutic agent against the tumor expressing the EML4-ALK fusionpolynucleotide for the EML4-ALK fusion polynucleotide positive patients.

(8) Anti-Tumor Test for Inhibitors of the EML4-ALK Fusion Polypeptideagainst v1 Expressing 3T3 Cells

3×10⁶ cells of v1 expressing 3T3 cells suspended in PBS were inoculatedsubcutaneously by injection to the back of 5 weeks old male BALB/c nudemice (Japan Charles River Inc.). After 7 days of the inoculation, theadministration of compound C, an inhibitor of the EML4-ALK fusionpolypeptide (Table 1 to 3), was initiated. The test was conducted in thesolvent group and compound C group, 4 animals per group. Compound C wasdissolved in a solvent composed of 10% 1-methyl-2-pyrrolidinone(SIGMA-ALDRICH Inc.)/90% polyethylene glycol 300 (Fluka Inc.) andadministered orally at the dose of 10 mg/kg. Administrations wereperformed once a day for 14 days, and body weight and tumor size weremeasured every other day. Tumor volume was calculated using thefollowing formula.[Tumor volume (mm³)]=[Tumor major axis (mm)]×[tumor minor axis(mm)]²×0.5

Assuming the tumor volume of the solvent group on the day of startingand the day of finishing administration was 100% inhibition and 0%inhibition, respectively, the inhibition rate of compound C wascalculated. The results indicated that compound C inhibited the growthof v1 expressing 3T3 cells (tumor) by 103%.

The antitumor effect of compound D was investigated by the similarprocedure with the following exceptions. The administration of thecompound D was started after 6 days of the inoculation and carried outonce a day for 10 days, and then the tumor size was measured. Compound Dinhibited the growth of v1 expressing 3T3 cells (tumor) by 101%.

(9) Kinase Inhibitory Effect by Repeated Administrations of theInhibitors of EML4-ALK Fusion Polypeptide to v1 Expressing 3T3 Tumor

The kinase inhibitory effect of compound C was observed by a similarmanner as in Example 8(8) with the following exceptions. 1×10⁶ cells ofv1 expressing 3T3 cells were inoculated and the administration ofcompound C was initiated after 13 day of the inoculation. The test wasconducted in the solvent group and the compound C group, 3 animals foreach group. The administrations were carried out once a day for 3 days.Animals were dissected 4 hours after the last administration, and thetumor was extirpated. Then, protein extracts were prepared from thetissues and immunoblotting was carried out using anti-phosphorylated ALKantibody. The results indicate that in the compound C group, tyrosineautophosphorylation of the EML4-ALK fusion polypeptide v1 in the tumorwas significantly decreased compared to the solvent group. From thisresult it was confirmed that the anti-tumor effect of compound C in theanimal model described above was based on the kinase inhibitory effectof the EML4-ALK fusion polypeptide v1 in the tumor.

Example 9 Detection of the EML4-ALK Fusion Polypeptide v1

A method for detecting the EML4-ALK fusion polypeptide v1 in cells wasconstructed as follows. The v1 expressing 3T3 cells and U-87MG cellswere cultured. After washing 3 times with PBS, cells were lysed with thelysis solution (Example 7(1)). To 4 mg of the supernatant obtained aftercentrifugation, anti-EML4 antibody (Cell Signaling Inc.) was added andreacted overnight at 4° C. Then, protein G beads (Protein G Sepharose 4Fast Flow; GE Healthcare Inc.) were added and immunoprecipitation wascarried out for 2 hours. After centrifugation, the precipitates werewashed 3 times with the washing solution (Example 7(1)) and suspended inan SDS dissolving solution. The supernatant was subjected toimmunoblotting using anti-ALK antibody. As the result, the EML4-ALKfusion polypeptide v1 was detected in the immunoprecipitates of v1expressing 3T3 cells but not detected in U-87MG cells. From the resultsdescribed above, it became possible to detect the presence of theEML4-ALK fusion polypeptide v1 in cancer cells and cancer tissuesexpressing the EML4-ALK fusion polypeptide v1 using anti-EML4 antibodyand anti-ALK antibody in combination, and it became clear that theEML4-ALK fusion polypeptide v1 positive cancer patients can bedetermined.

Example 10 Analysis of the EML4-ALK Fusion Polypeptide v2

(1) Construction of an Expression Vector of EML4-ALK Fusion Polypeptidev2

By consulting the non-patent document 8, using the full length EML4polynucleotide cloned to pT7Blue as a template, an oligonucleotiderepresented by the SEQ ID NO: 129 which was provided with the cleavagesequence of restriction enzyme HindIII at the 5′ terminus side of thestart codon ATG of the EML4 gene and an oligonucleotide represented bySEQ ID NO: 130 which was designed to contain the cleavage sequence ofXhoI present in the EML4 gene as primers, and a DNA polymerase (PyrobestDNA polymerase; Takara Bio Inc.), PCR (25 cycles of 94° C. fro 20seconds, 60° C. for 30 seconds and 72° C. for 1 minute) was carried outto obtain a PCR product of 238 bp. Using this PCR product as a template,an oligonucleotide represented by SEQ ID NO: 131 which was provided withthe cleavage sequence of restriction enzyme XhoI at the 5′ terminus andthe aforementioned oligonucleotide represented by SEQ ID NO: 130 asprimers, PCR was carried out in the same conditioned as described aboveto obtain a PCR product of 247 bp. This product was digested withrestriction enzyme XhoI and ligated with restriction enzyme XhoIdigested pCR2.1-TOPO vector in which the EML4-ALK fusion polynucleotidev2 produced in Example 4(1) was cloned to produce a vector in which thecleavage sequence of restriction enzyme HindIII was integrated at the 5′terminus side of the start codon of the EML4-ALK fusion polynucleotidev2 (EML4-ALKv2/pCR2.1).

EML4-ALKv2/pCR2.1 was digested with restriction enzyme HindIII, theEML4-ALK fusion polynucleotide v2 was excised, the both termini wereblunted, an adapter (EcoRI-NotI-BamHI adaptor; Takara Bio Inc.) was thenligated to both termini, and the fragment was inserted to the EcoRI siteof a retrovirus vector pMXS. This was designated as EML4-ALKv2/pMXS.

Also, EML4-ALKv2/pCR2.1 was digested with restriction enzyme HindIII andXbaI, the EML4-ALK fusion polynucleotide v2 was excised and inserted tothe HindIII/XbaI site of an expression vector pcDNA3.1/Zeo (InvitrogenInc.) which was modified so that the FLAG tag was attached to theN-terminus on expression to produce an expression plasmid for EML4-ALKfusion polypeptide v2 to which the FLAG tag was attached to the Nterminus (FLAG-EML4-ALKv2/pcDNA3).

(2) Confirmation of an Intracellular Autophosphorylation Activity of theEML4-ALK Fusion Polypeptide v2 and Screening for the Substances whichInhibit its Activity

To 293EBNA cells (Invitrogen Inc.) seeded in collagen I coated 24 wellplates (IWAKI; Asahi techno glass corp.) at 1×10⁵ cells per well in DMEMmedium containing 10% fetal bovine serum, 100 ng ofFLAG-EML4-ALKv2/pcDNA3 (Example 10(1) or pcDNA3 (blank vector) as acontrol was introduced using a transfection reagent (Lipofectamin2000;Invitrogen Inc.). After culturing for 20 hours, compound C or D each wasadded, and the culture was incubated for 4 hours and then cells wererecovered. Expression of EML4-ALK fusion polypeptide v2 and tyrosinephosphorylation level were measured by immunoblotting using anti-ALKantibody and anti-phosphorylated ALK antibody.

As the results, in the immunoblot using anti-ALK antibody, a band wasconfirmed at the location of about 160 kDa where the EML4-ALK fusionpolynucleotide v2 was expected to be present, and it was demonstratedthat the amount of protein expression itself of the EML4-ALK fusionpolynucleotide v2 was almost constant in all the samples. In addition, aband was confirmed at the same location in the immunoblot usinganti-phosphorylated ALK antibody. The amount of phosphorylation wascalculated by quantitating in a similar manner as in Example 7(2). Theinhibition rate of phosphorylation by a compound was calculated from theamount of phosphorylation when the compound was added, assuming thevalue when no compound was added (the solvent of the compound, DMSO, wasadded) was 0% inhibition rate, and the value when the empty vector,pcDNA3, was introduced was 100% inhibition rate. The results indicatethat each compound clearly inhibited the kinase activity of the EML4-ALKfusion polynucleotide v2 in 293EBNA cells (Table 6). Compound C and Dcould be selected as substances which inhibited the activity of theEML4-ALK fusion polypeptide v2 by 50% or more at a concentration of 0.1μM or less. It is confirmed that screening for the substances inhibitingthe activity of the polypeptide of the present invention (cell typescreening) can be performed using the EML4-ALK fusion polypeptide v2 aswell as the EML4-ALK fusion polypeptide v1.

TABLE 6 Autophosphorylation Final inhibition Compound concentration(293EBNA cells) C 10 nM 90% D 10 nM 95%(3) Investigation of Transformability and Tumorigenicity of EML4-ALKFusion Polypeptide v2(3-1) Focus Formation Assay

By a similar manner described as in Example 6(1), the focus formingability of EML4-ALK fusion polynucleotide v2 was investigated usingEML4-ALKv2/pMXS (Example 10(1)). As a result, transformed foci of 3T3cells were observed 21 days after transfection.

(3-2) Tumorigenicity in Nude Mice

FLAG-EML4-ALKv2/pcDNA3 (Example 10(1)) was transfected into 3T3 cellsusing a transfection regent (FuGENE HD; Roche Diagnostics Inc.)according to the attached instruction. The EML4-ALK fusionpolynucleotide v2 stably expressing 3T3 cells were established byresistance to 80 μg/ml zeocin. The expression of EML4-ALK fusionpolynucleotide v2 in the 3T3 cells was confirmed by immunoblotting usinganti-ALK antibody and anti-phosphorylated ALK antibody. The 3T3 cells inwhich EML4-ALK fusion polypeptide v2 is expressed are designated as thev2 expressing 3T3 cells. The v2 expressing 3T3 cells were inoculatedsubcutaneously to 4 weeks old male BALB/c nude mice (Japan Charles RiverInc.) at 2×10⁶ cells/mouse and observed for 15 days. As in the case ofthe v1 expressing cells (the lower section of the FIG. 3), it turned outthat tumor was also formed in EML4-ALK fusion polypeptide v2 expressing3T3 cells. The tumor formation number was 4 among 4.

From above results, it was confirmed that the EML4-ALK fusionpolynucleotide v2, like the EML4-ALK fusion polynucleotide v1, is alsoan oncogene which codes for the polypeptides having the transformabilityand tumorigenicity to 3T3 cells.

1. A method for detecting a fusion gene of EML4 gene and ALK gene, comprising the step of detecting the presence of the polynucleotide encoding a polypeptide in a sample obtained from a test subject, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2. 